Deteksi Gen CehA Pengkode Enzim Karbofuran Hidrolase pada Bacillus cereus Strain HS06

Abstract

The cehA gene is one of the genes encoding the carbofuran hydrolase (KH) enzyme, which plays a role in the biodegradation of the insecticide carbofuran. This study aimed to detect the presence of the cehA gene in Bacillus cereus strain HS06 as one of the carbofuran-degrading bacteria. Gene detection was carried out using four cehA-specific primers. Three primers (cehA1, cehA2, and cehA3) were designed using Primer3 software based on the complete genomes of three Bacillus cereus strains possessing genes encoding the CehA/McbA metallohydrolase enzyme registered in GenBank, while one primer (cehA4) was obtained from the literature. Primer cehA1 was designed based on the complete genome of B. cereus strain SIN1.2 with a target amplicon size of 506 bp, primer cehA2 based on the complete genome of B. cereus strain D8_B_47 with a target of 877 bp, and primer cehA3 based on the complete genome of B. cereus strain D5_B_69 with a target of 552 bp. Genomic DNA of B. cereus strain HS06 was isolated and amplified using these four primers. The PCR results showed that the obtained amplification bands did not correspond to the target sizes of the cehA gene. The clearest fragment produced by primer cehA2 was subsequently sequenced. BLAST analysis showed that the sequence obtained was not homologous to the cehA gene but exhibited a higher similarity to the genome of Bacillus tropicus strain PCr-6. Phylogenetic analysis using the Neighbor-Joining method indicated that the putative cehA gene of Bacillus cereus strain HS06 did not exhibit close phylogenetic relatedness to all reference sequences, as evidenced by its position on a separate branch with a relatively long branch length and an evolutionary distance of approximately 0.20, indicating a high level of nucleotide divergence and its incongruence with the reference cehA genes. Based on the PCR results, sequence analysis, and phylogenetic analysis, it can be concluded that the cehA gene was not detected in Bacillus cereus strain HS06.