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Chitinases, a glycosyl hydrolase family 18 members, have a wide distribution in both prokaryotes and eukaryotes, including humans. Regardless of the absence of endogenous chitin polymer, various chitinases and chitinase-like proteins (CLPs) have been reported in mammals. However, several other carbohydrate polymers, such as hyaluronic acid and heparan sulfate, show structural similarities with chitin, which could be a potential target of chitinase and CLPs. Heparan sulfate is part of the integral membrane proteins and involves in cell adherence and migration. Hence, to demonstrate the effect of chitinase on cancer cell progression, we selected two chitinases from Serratia marcescens , ChiB and ChiC, which function as exo- and endo-chitinase, respectively. The ChiB and ChiC proteins were produced recombinantly by cloning chiB and chiC genes from Serratia marcescens . The cell viability of the Michigan Cancer Foundation-7 (MCF-7) cells was studied using different concentrations of the purified recombinant proteins. Cell viability assay was performed using 3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide and water-soluble tetrazolium salt, and the effect of ChiB and ChiC on cell proliferation was studied by clonogenic assay. The cell migration study was analysed by wound healing, transwell migration, and invasion assays. Cell cycle analysis of propidium iodide–stained cells and cell proliferation markers such as pERK1/2, pAKT, and SMP30 were also done. It was observed that both ChiB and ChiC were able to impede cell viability, cell migration, and invasion significantly. These observations and our in silico molecular docking analysis suggest that ChiC is a potential anticancer agent and is more efficient than ChiB. Since the ChiC is able to inhibit both cancer cell proliferation and migration, it could be a potential candidate for the treatment of metastatic cancer. Graphical Abstract

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.

An acid-active exo/endo-chitinase; comprising a GH18 catalytic domain and substrate insertion domain; originating from the thermophilic filamentous fungus Rasamsonia emersonii, was expressed in Pichia pastoris. In silico analysis including phylogenetic analysis, and recombinant production, purification, biochemical characterisation, and industrial application testing, was carried out. The expressed protein was identified by SDS-PAGE as a smear from 56.3 to 125.1 kDa, which sharpens into bands at 46.0 kDa, 48.4 kDa and a smear above 60 kDa when treated with PNGase F. The acid-active chitinase was primarily a chitobiosidase but displayed some endo-chitinase and acetyl-glucosamidase activity. The enzyme was optimally active at 50 °C, and markedly low pH of 2.8. As far as the authors are aware, this is the lowest pH optima reported for any fungal chitinase. The acid-active chitinase likely plays a role in chitin degradation for cell uptake in its native environment, perhaps in conjunction with a chitin deacetylase. Comparative studies with other R. emersonii chitinases indicate that they may play a synergistic role in this. The acid-active chitinase displayed some efficacy against non-treated substrates; fungal chitin and chitin from shrimp. Thus, it may be suited to industrial chitin hydrolysis reactions for extraction of glucosamine and chitobiose at low pH.

A new chitinase gene, cloned from the biocontrol Chaetomium globosum W7, was designated Cgchi18. Recombinant protein Cgchi18 with 535 amino acids was expressed in Escherichia coli , and purified by means of a column—free purification method relying on split intein, achieving a 12.39—fold purification and a 15.61% recovery yield. The maximum activity of this approximately 60-kDa protein was observed at 45 °C and pH 5.0. Cgchi18 was activated by Mg 2+ and Ba 2+ , but inhibited by Mn 2+ , Co 2+ , Cu 2+ , Zn 2+ , Ag + and Hg 2+ . Cgchi18 showed high substrate specificity, only hydrolyzing β-1,4-glycoside bond in chitin and its derivatives, to liberate disaccharides or trisaccharides. For the degradation of colloidal chitin under optimal conditions, Vmax and Km of Cgchi18 were calculated as 8.05 μmol/min/mg and 3.18 mg/mL, respectively. Additionally, it exhibited antifungal activity and could have a degrading effect on the spread of hyphae of pathogenic fungi. In conclusion, the chitinase Cgchi18 identified from C. globosum has potential for industrial and agricultural applications.

Abstract Acidic chitinase (Chia) digests the chitin of insects in the omnivorous stomach and the chitinase activity in carnivorous Chia is significantly lower than that of the omnivorous enzyme. However, mechanistic and evolutionary insights into the functional changes in Chia remain unclear. Here we show that a noninsect-based diet has caused structural and functional changes in Chia during the course of evolution in Carnivora. By creating mouse-dog chimeric Chia proteins and modifying the amino acid sequences, we revealed that F214L and A216G substitutions led to the dog enzyme activation. In 31 Carnivora, Chia was present as a pseudogene with stop codons in the open reading frame (ORF) region. Importantly, the Chia proteins of skunk, meerkat, mongoose, and hyena, which are insect-eating species, showed high chitinolytic activity. The cat Chia pseudogene product was still inactive even after ORF restoration. However, the enzyme was activated by matching the number and position of Cys residues to an active form and by introducing five meerkat Chia residues. Mutations affecting the Chia conformation and activity after pseudogenization have accumulated in the common ancestor of Felidae due to functional constraints. Evolutionary analysis indicates that Chia genes are under relaxed selective constraint in species with noninsect-based diets except for Canidae. These results suggest that there are two types of inactivating processes in Carnivora and that dietary changes affect the structure and activity of Chia.

Chitinases, a group of glycosyl hydrolases (GHs), catalyze the degradation of chitin by releasing N-acetylglucosamine subunits. GHs can be found in all three domains of life. Among the three GH families (GH18, GH19, and GH20), GH18 chitinases are the most conserved and extensively studied. These enzymes have been implicated in nutrition, immune modulation, invasion, and virulence across diverse pathogens. In some parasitic protists, GH18 chitinases are essential for transmission. However, in kinetoplastids, including Leishmania spp., these enzymes remain poorly characterized. This study aimed to identify and characterize chitinase across kinetoplastids, with a particular focus on GH18 chitinase in Leishmania braziliensis , the causative agent of muco-cutaneous leishmaniasis. A bioinformatic pipeline was implemented to retrieve and annotate chitinase genes from multiple databases. Catalytic domains and subcellular localization were identified using dedicated computational tools. Phylogenetic relationships were reconstructed in MEGA12 using maximum likelihood and neighbor-joining methods. RNA-seq data were analyzed to evaluate stage-specific expression. Structural models of L. braziliensis GH18 chitinase (Lbr_ChGH18) were created with AlphaFold and subsequently refined. We identified GH18 chitinases genes across Leishmania species and other kinetoplastids, together with GH19 and GH20 chitinase genes. Lbr_ChGH18 was detected across all life-cycle stages, with peak levels in amastigotes. Docking analyses identified closantel, argifin, and argadin as potential inhibitors of Lbr_ChGH18. Epitope prediction revealed conserved B- and T-cell epitopes in GH18 chitinases from Old and New World Leishmania , capable of binding multiple HLA class I and II molecules, highlighting their potential as diagnostic and vaccine candidates. The discovery of GH20 chitinase-like genes in Trypanosoma species offers new insights into the evolution and functional diversification of GHs in kinetoplastids. The high conservation and expression of GH18 chitinases underscore their potential as promising targets for drug and vaccine development against leishmaniasis.

Chitin derivatives, chitosan and substituted chito-oligosaccharides have a wide spectrum of applications ranging from medicine to cosmetics and dietary supplements. With advancing knowledge about the substrate-binding properties of chitinases, enzyme-based production of these biotechnologically relevant sugars from biological resources is becoming increasingly interesting. Fungi have high numbers of glycoside hydrolase family 18 chitinases with different substrate-binding site architectures. As presented in this review, the large diversity of fungal chitinases is an interesting starting point for protein engineering. In this review, recent data about the architecture of the substrate-binding clefts of fungal chitinases, in connection with their hydrolytic and transglycolytic abilities, and the development of chitinase inhibitors are summarized. Furthermore, the biological functions of chitinases, chitin and chitosan utilization by fungi, and the effects of these aspects on biotechnological applications, including protein overexpression and autolysis during industrial processes, are discussed in this review. [PUBLICATION ABSTRACT]

Anthracnose caused by Colletotrichum acutatum is one of the most devastating fungal diseases of pepper (Capsicum annuum L.). The utilization of chitin-binding proteins or chitinase genes is the best option to control this disease. A chitin-binding domain (CBD) has been shown to be crucial for the innate immunity of plants and activates the hypersensitive response (HR). The CaChiIII7 chitinase gene has been identified and isolated from pepper plants. CaChiIII7 has repeated CBDs that encode a chitinase enzyme that is transcriptionally stimulated by C. acutatum infection. The knockdown of CaChiIII7 in pepper plants confers increased hypersensitivity to C. acutatum, resulting in its proliferation in infected leaves and an attenuation of the defense response genes CaPR1, CaPR5, and SAR8.2 in the CaChiIII7-silenced pepper plants. Additionally, H2O2 accumulation, conductivity, proline biosynthesis, and root activity were distinctly reduced in CaChiIII7-silenced plants. Subcellular localization analyses indicated that the CaChiIII7 protein is located in the plasma membrane and cytoplasm of plant cells. The transient expression of CaChiIII7 increases the basal resistance to C. acutatum by significantly expressing several defense response genes and the HR in pepper leaves, accompanied by an induction of H2O2 biosynthesis. These findings demonstrate that CaChiIII7 plays a prominent role in plant defense in response to pathogen infection.

Acidic chitinase (Chia) degrades chitin, a structural polysaccharide in insect exoskeletons, and plays important roles in omnivorous and insectivorous mammals and birds. In birds, gene duplications have generated multiple Chia paralogs with functional divergence, but the molecular basis for this diversification remains unclear. Here, we characterized three chicken Chia paralogs (Chia1–3) and identified distinct pH-dependent enzymatic profiles. Chia1 is enzymatically inactive but was captured by chitin-affinity resin despite lacking a canonical chitin-binding domain, suggesting residual substrate interaction through the catalytic domain or a non-catalytic role. Chia2 exhibits maximal activity at pH 2.0, whereas Chia3 peaks at pH 5.0 and displays broader activity. Exon swapping and site-directed mutagenesis identified residues 104 (Ala in Chia2, Asp in Chia3) and 269 (His vs. Asn) as key contributors to pH-dependent activity differences. Reciprocal substitutions shifted pH profiles accordingly. Structural modeling and computational pKa predictions suggested that D213 and residue 269 may function as a pKa-regulating module influencing catalytic ionization. Comparative sequence analysis revealed lineage-specific conservation of these residues, consistent with adaptive divergence. Our findings show that limited amino acid substitutions can markedly modify pH-dependent enzymatic activity, providing mechanistic insight into how local residue variation contributes to the functional diversification of duplicated genes.

Chitinases are glycosyl hydrolase enzymes that break down chitin, an integral component of fungal cell walls. Bacteria such as Bacillus subtilis and Serratia marcescens produce chitinases with antifungal properties. In this study, we aimed to generate hybrid chitinase enzymes with enhanced antifungal activity by combining functional domains from native chitinases produced by B. subtilis and S. marcescens . Chitinase genes were cloned from both bacteria and fused together using overlap extension PCR. The hybrid constructs were expressed in E. coli and the recombinant enzymes purified. Gel electrophoresis and computational analysis confirmed the molecular weights and isoelectric points of the hybrid chitinases were intermediate between the parental enzymes. Antifungal assays demonstrated that the hybrid chitinases inhibited growth of the fungus Fusarium oxysporum significantly more than the native enzymes and also showed fungicidal activity against Candida albicans, Alternaria solani, and Rhizoctonia solani . The results indicate that hybrid bacterial chitinases are a promising approach to engineer novel antifungal proteins. This study provides insight into structure–function relationships of chitinases and strategies for generating biotherapeutics with enhanced bioactive properties. These hybrid chitinases result in a more potent and versatile antifungal agent. Graphical Abstract