Isolasi, Sekuensing, dan Kloning Gen pcbC Pengkode Enzim Isopenicillin N Synthase Family Oxygenase dari Serratia plymuthica UBCF_13 pada Escherichia coli

Fauzan Syarif, Nursyafi (2024) Isolasi, Sekuensing, dan Kloning Gen pcbC Pengkode Enzim Isopenicillin N Synthase Family Oxygenase dari Serratia plymuthica UBCF_13 pada Escherichia coli. Diploma thesis, Universitas Andalas.

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Abstract

Bacterial infections are a leading cause of global mortality and a priority health issue identified by WHO. Antibiotics are commonly used to treat these infections, but excessive use has led to resistance in many organisms. Developing semisynthetic penicillin is one approach to overcoming resistance due to its better antimicrobial activity. Isopenicillin N synthase (IPNS) is crucial for the biosynthesis of semi-synthetic penicillin, as it catalyzes the oxidative condensation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (LLD-ACV) to isopenicillin N. Through genome mining studies of Serratia plymuthica (S. plymuthica) UBCF_13, the presence of the pcbC gene encoding isopenicillin N synthase family oxygenase (IPNSfo) was revealed. Therefore, in order to provide a low-cost and high-performance IPNS enzyme, this study aims to isolate, sequence, and clone the pcbC gene from S. plymuthica UBCF_13 in Escherichia coli (E. coli). To achieve this objective, the following tasks were carried out: Isolation of the pcbC gene using PCR with specific primers, sequencing, bioinformatics analysis for further enzyme characterization, and cloning the pGEM_pcbC vector into E. coli. Electrophoresis confirmed successful gene isolation, with the optimal annealing temperature for the pcbC gene-specific primer at 64.4°C. Colony PCR verified the presence of pGEM_pcbC in transformant bacteria. Sequencing analysis shows that the Consensus_sequence_pcbC has a percent identity of 100% (919/919), Gaps 0% (0/919), query cover 100%, and phylogenetic distance of 0% with the genome of S. plymuthica UBCF_13. Bioinformatics analysis showed successful enzyme structure modeling with I-TASSER. Additionally, domain analysis identified three main domains in the IPNS enzyme: DIOX_N, IPNS-like_FE2OG_OXY, and Oxoglu/Fe_dep_dioxygenase. These findings demonstrate the successful isolation, sequencing, and cloning of the pcbC gene from S. plymuthica UBCF_13 in E. coli.

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