CLONING OF POLY-(R)-3-HYDROXYBUTYRATE BIOSYNTHESIS GENES FROM BACILLUS THURINGIENSIS TH-01

• Main Author: Sabiqoh, Zuhdina
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/47523
• Institution: Institut Teknologi Bandung

Poly-(R)-3-hydroxybutyrate (PHB) is a polyhydroxyalkanoate (PHA) derivative bioplastic material that can be synthesized biologically by bacteria under growth conditions with poor key nutrients but rich in carbon sources. Bacillus thuringiensis TH-01 isolated from thermite nest was identified to accumulate PHB with a yield of 9.13%. PHB biosynthesis in Bacillus involves three enzymes encoded by four different genes, namely phaA (encoding acetyl-CoA acetyltransferase), phaB (encoding acetoacetyl-CoA reductase), also phaC and phaR (encoding PhaC and PhaR subunits of PHA synthase). This study aims to clone the genes that involved in the biosynthesis of PHB from Bacillus thuringiensis TH-01 and predict the tertiary structure of the encoded enzymes. Gene isolation was carried out using Polymerase Chain Reaction (PCR) technique with Bacillus thuringiensis TH-01chromosome as template, and the obtained amplicon was ligated to pGEM-T Easy vector which then transformed into E. coli TOP10 host. Based on the results of re-PCR and nucleotide sequence analysis, phaA and phaRBC were confirmed to be in the recombinant plasmid pGEM-Bt-phaA and pGEM- Bt-phaRBC. The length of phaA cloned gene is 1092 bp, while phaRBC cloned gene cluster is 2937 bp, which is comprised of 744 bp phaB, 1086 bp phaC, and 483 bp phaR that encode for PhaB, PhaC, and PhaR proteins. Bioinformatics analysis and tertiary structure prediction of PhaA, PhaB, and PhaC based on the sequence of the translated genes using I-TASSER give results of PhaA, PhaB, and PhaC structues with 363, 247, and 361 amino acids residues respectively. PhaA and PhaB were identified to have a dominant structure of ?/? fold, The catalytic residues in PhaA are Cys83, His317, and Gly349 whereas the catalytic residues in PhaB are Ser143, Tyr156, and Lys160. Based on the alignment of predicted PhaC with PHA synthase, the PhaC residues of Cys151, Asp306, dan His335 are overlapped with the catalytic residues of PHA synthase from Chromobacterium sp. USM2.