Efficient expression of truncated Taq DNA polymerase in Escherichia coli and its application in mitochondrial cytochrome b-targeted PCR for halal authentication.

Halal authentication using the polymerase chain reaction (PCR) technique relies on amplifying specific DNA sequences derived from non-halal sources, which requires thermostable enzymes such as Taq DNA polymerase. Currently, most commercial halal detection kits in Indonesia, as a Muslim-majority country, rely on imported Taq DNA polymerase enzymes for halal detection kits, resulting in limited access. In this study, the production of in-house recombinant Taq DNA polymerase (TaqPol) expressed in Escherichia coli BL21 Star (DE3) containing plasmid pD871 was optimized, and the TaqPol was applied as an enzyme in a halal detection kit in optimized condition. The optimization of expression parameters, including post-induction incubation time, IPTG concentration, and cell density (OD600), was carried out systematically. The optimum conditions obtained with maximum TaqPol production were a post-induction incubation time of 18 h, an IPTG concentration of 0.4 mM, and an OD600 of 0.6. Under these optimized conditions, the enzyme yield increased by approximately 145%, resulting in 10 mg of purified protein per liter of culture with a total activity of 2350 U. SDS-PAGE analysis confirmed a single band at approximately 63 kDa. To enhance the sensitivity and specificity of the halal detection, the reverse primer used was based on the porcine mitochondrial cytochrome b (cyt b) gene. This approach enables the detection of porcine DNA in both fresh and processed meat products. In addition, the PCR performance optimization was carried out by optimizing the annealing temperature and enzyme unit concentration. This optimization was then applied to various non-halal test samples, and the results showed that an annealing temperature of 65 °C and an enzyme concentration of 0.5 U per 25 µL reaction produced consistent and specific DNA amplification in various sample types. These results confirmed the suitability of the optimized parameters for the halal detection kit using the PCR technique with the TaqPol. Comparative analysis with imported Taq DNA polymerase showed comparable amplification performance of band intensity and clarity. Overall, this study shows that the TaqPol has strong potential to be used as an independent halal detection kit in Indonesia, thus supporting the independence of halal detection kits in Indonesia.