Recombinant TP-84 Bacteriophage Glycosylase-Depolymerase Confers Activity against Thermostable Geobacillus stearothermophilus via Capsule Degradation.

The TP-84 bacteriophage, which infects Geobacillus stearothermophilus strain 10 ( G. stearothermophilus ), has a genome size of 47.7 kilobase pairs (kbps) and contains 81 predicted protein-coding ORFs. One of these, TP84_26 encodes a putative tail fiber protein possessing capsule depolymerase activity. In this study, we cloned the TP84_26 gene into a high-expression Escherichia coli ( E. coli ) system, modified its N-terminus with His-tag, expressed both the wild type gene and His-tagged variant, purified the recombinant depolymerase variants, and further evaluated their properties. We developed a direct enzymatic assay for the depolymerase activity toward G. stearothermophilus capsules. The recombinant TP84_26 protein variants effectively degraded the existing bacterial capsules and inhibited the formation of new ones. Our results provide insights into the novel TP84_26 depolymerase with specific activity against thermostable G. stearothermophilus and its role in the TP-84 life cycle. The identification and characterization of novel depolymerases, such as TP84_26, hold promise for innovative strategies to combat bacterial infections and improve various industrial processes.