GENE CLONING AND IN SILICO STUDY OF ENZYMES IN POLY-(R)-3-HYDROXYBUTYRATE BIOSYNTHESIS FROM BACILLUS CEREUS TH-02

• Main Author: Raeh Rizkia, Pidinia
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/71603
• Institution: Institut Teknologi Bandung

Poly-(R)-3-hydroxybutyrate (PHB) is a polyhydroxyalkanoate (PHA) bioplastic which can be degraded and is most commonly synthesised by microorganisms in response to physiological stress conditions. PHB is a biopolymer that accumulates as intracellular granules and functions as an energy reserve for these microorganisms. PHB biosynthesis in Bacillus cereus involves three enzymes, namely acetyl-CoA C-acetyltransferase (encoded by phaA gene), acetoacetyl-CoA reductase (encoded by phaB gene) and PHA synthase (encoded by phaR and phaC genes). Bacillus cereus TH-02 is a PHB-producing bacterium isolated from thermite. This study aims to confirm the Bacillus cereus strain by 16S ribotyping, isolate the phaA gene and phaRBC gene cluster by Polymerase Chain Reaction (PCR) technique using primers based on similar genes from Bacillus thuringiensis, clone these genes into pGEM-T vector with host cells E. coli TOP'10, determine the nucleotide sequence, and deduce the primary structure of the protein using Expasy. Furthermore, tertiary structure prediction and prediction of the catalytic residues of the PhaA, PhaB, and PhaC proteins were carried out. The ribotyping results confirmed that the bacteria used was Bacillus cereus. The cloned genes have sizes of 1092bp, 744bp, 483bp, and 1087bp for phaA, phaB, phaR, and phaC, respectively. The results of the prediction of catalytic residues using I-TASSER showed the role of Asn115, Tyr156 and Lys160 in phaB. The catalytic residues have been identified in a similar gene from Bacillus anthracis. On the other hand, the alignment of PhaC with polyhydroxybutyrate synthase from Cupriavidus necator showed catalytic residues Cys151, Asp306, and His335 in PhaC. The success of this cloning is expected to increase PHB production using recombinant clones.