Journal Article

Preparation and enzymatic activity analysis of rChiA-DP derived from the Bacillus proteolyticus IMH/B-1 Strain isolated from Dermacentor nuttalli

Yila, Geqi, Shah, Syed Qaswar Ali, Hassan, Muhammad Adeel, Teri, Gele, Liu, Yuxin, Sua, Rina, Geri, Letu, Liu, Fang, Huge, Jile

2025

Overview

This study aimed to clone and express chitinase genes from Bacillus proteolyticus strains and characterize the enzymatic properties of recombinant enzymes. Bacillus proteolyticus was isolated from the body of Dermacentor nuttalli and renamed IMH/B-1. Chitin-degrading enzymes were screened via clear zone assay and PCR. The chitinase A gene ( ChiA ) was successfully cloned, and the recombinant plasmid pET28a-rChiA-DP (Dermatestor-derived Protein) was constructed. Recombinant chitinase protein (rChiA-DP) was expressed in Escherichia coli BL21 using IPTG induction and purified by nickel-nitrilotriacetic acid (Ni–NTA) affinity chromatography. Bioinformatic tools were used to predict the rChiA-DP protein sequences, analyse its enzyme family classification, and identify key amino acid residues in its catalytic domain. The enzymatic activity of rChiA-DP, along with its nematode resistance and antifungal effects on Caenorhabditis elegans ( C. elegans ) and fungi ( Aspergillus sp.), was assessed under varied temperatures, pH, metal ions, salt concentrations and substrates. The amino acid sequence of the rChiA-DP contains a chitin-binding domain (CBD) (substrate binding), a fibronectin type III domain (FN3)(structural stability), and a catalytic domain with a typical TIM-barrel molecular structure (catalytic scaffold). 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 Ba 2+ , 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 Cu 2+ , Ni 2+ , Fe 3+ , Zn 2+ and Mn 2+ as well as SDS, DMSO, Tween 20/80 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 (7.53 ± 0.86 U/mL). However, it exhibited no degradation activity on chitosan and tick surface. Compared with the heat-inactivated control group‌ and the s-buffer group, the rChiA-DP treatment significantly reduced the survival rate of C. elegans by 50.4% vs. heat-inactivated control ( P  < 0.01), indicating a potential antiparasitic effect. However, it showed no significant antifungal activity against fungi such as Aspergillus niger or Aspergillus flavus, and the diameter of the inhibition zone was not significantly larger than that of the negative control ( P  > 0.05). This study successfully prepared tick-derived rChiA-DPs and evaluated their enzymatic activity and anti-nematodal activity, providing enzymatic basis for the design of biopesticides targeting insect cuticle.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 631/208 / 631/326 / Affinity chromatography / Alcohol / Amino Acid Sequence / Amino acids / Animals / Antifungal activity / Antifungal Agents - pharmacology / Antiparasitic agents / Bacillus / Bacillus - enzymology / Bacillus - genetics / Bacillus - isolation & purification / Bacteria / Bacterial Proteins - chemistry / Bacterial Proteins - genetics / Bacterial Proteins - metabolism / Biopesticides / Biotechnology / Caenorhabditis elegans / Caenorhabditis elegans - drug effects / Chitin / Chitin - metabolism / Chitinase / Chitinases - chemistry / Chitinases - genetics / Chitinases - isolation & purification / Chitinases - metabolism / Chitinases - pharmacology / Chitosan / Cloning, Molecular / Dermacentor - microbiology / Dermacentor nuttalli / DP protein / E coli / Enzymatic activity / Enzymes / Epicuticle / Escherichia coli - genetics / Ethanol / Fibronectin / Fungi / Gene amplification / Humanities and Social Sciences / Hydrogen-Ion Concentration / Isopentyl alcohol / Laboratories / Metal concentrations / Metal ions / Molecular weight / multidisciplinary / Nematodes / Nitrilotriacetic acid / Organic solvents / Recombinant chitinase / Recombinant Proteins - chemistry / Recombinant Proteins - genetics / Recombinant Proteins - isolation & purification / Recombinant Proteins - metabolism / Recombinant Proteins - pharmacology / Science / Science (multidisciplinary) / Zinc