Molecular Engineering of the Thioredoxin (trxA) Gene in Chryseobacterium proteolyticum Enhanced its Keratinase Bioconversion of Chicken Feathers.

<b>Background and Objective:</b> Keratinases are gaining traction in green technology and the bio-economy due to their ability to convert keratinous biomass into valuable products, such as animal feed. However, improving their output remains challenging. This study investigated genetic engineering approaches to increase keratinase production. <b>Materials and Methods:</b> The genome of the keratinolytic bacterium, <i>C. proteolyticum,</i> was sequenced using the Ion GeneStudio<sup>™</sup> S5 Prime System, covering 2,484,843 reads, and sequence reads generated were assembled using SPAdes 3.13.0. The quality of the reads was assessed with QUAST V4.1, and the obtained sequences were annotated with Prokka 1.14.6, which revealed that trxA is associated with keratinolytic activity. The nucleotide sequence obtained was uploaded to the NCBI database and was assigned the accession number JBJLWK000000000. The <i>trxA</i> gene was amplified with primers from NCBI Primer-BLAST, cloned into a p-GEM<sup>®</sup>-T Easy vector, and transformed into <i>E. coli</i> JM109, and expressed using IPTG and SDS-PAGE analysis. The effects of pH and temperature on enzyme production were studied, and the recombinant strain's thermostability and tolerance to commercial laundry detergents were assessed. <b>Results:</b> The gene was successfully cloned and expressed, with SDS-PAGE showing a clear band at 11 kDa, matching trxA's molecular weight. The pGEM-trxA construct demonstrated high keratinase activity across various pH and temperature conditions. Additionally, the recombinant strain showed excellent thermostability and detergent tolerance. <b>Conclusion:</b> These findings highlight the potential of recombinant technology to boost keratinase yields and support the sustainable recycling of keratinous waste.