Identification of rChiA DP enzyme activity in the tick-derived Bacillus IMH/B-1 strain and its enzymatic analysis

Background: This study aimed to clone and express chitinase genes from Bacillus proteolyticus strains and investigate the enzymatic properties of recombinant enzymes. Materials and methods: Bacillus proteolyticus was isolated from the body of Dermacentor nuttalli and renamed IMH/B-1. Enzymes capable of degrading chitin were identified via the transparent circle method and PCR. The chitinase A (ChiA) gene was successfully cloned, and the recombinant plasmid pET28a-rChiA DP was constructed. Recombinant chitinase protein (rChiA DP) was produced through IPTG-induced expression in Escherichia coli BL21 and purified via nickel column affinity chromatography. Bioinformatic tools were used to predict the rChiA-DP protein sequences, analyse its enzyme family classification, and identify key amino acid residues involved in substrate interactions. The enzymatic activity of rChiA-DP, along with its antiparasitic and antifungal effects on Caenorhabditis elegans (C. elegans) and fungi (Aspergillus sp.), was evaluated under various conditions, such as various temperatures, pH values, metal ions, salt concentrations and substrates. Results: The amino acid sequence of the rChiA DP contains a chitin-binding domain (CBD), a fibronectin type III domain (FN3), and a catalytic domain with a typical TIM barrel molecular structure. SDS‒PAGE analysis revealed that the molecular weight of the rChiA DP was approximately 74.6 kDa, which is consistent with the theoretical predictions. The optimal conditions for rChiA-DP enzyme activity were 40°C and pH 7.0. Enzyme activity was significantly enhanced by 10 mM Ba²⁺, Tris, K⁺, and Li⁺. Organic solvents such as methanol, ethanol, isopropanol, and isoamyl alcohol (10% concentration) also increased the activity. Conversely, positive metal ions such as Cu²⁺, Ni²⁺, Fe³⁺, Zn²⁺ and Mn²⁺ as well as SDS, DMSO, Tween 80, and Tween 20 significantly inhibited the activity of the rChiA DP. rChiA DPs demonstrated varying degrees of decomposition activity against substrates such as colloidal chitin, chitin powder, nematode eggs, nematodes, shrimp shells, and tick eggs, with the highest activity observed for colloidal chitin (7.529±0.859 U/ml). However, it exhibited no activity against chitosan or ticks. Compared with the control group (high-temperature inactivation treatment) and the s-buffer group, the rChiA DP treatment significantly reduced the survival rate of C. elegans by 50.4% and 55.2%, indicating a potent antiparasitic effect (P<0.01). However, it showed no significant antifungal activity against fungi such as Aspergillus niger and Aspergillus flavus. Conclusions: This study successfully prepared tick-derived rChiA DPs and evaluated their enzymatic activity and antiparasitic effects, providing a foundation for the development of new biological pesticides.