Introduction

Fungal diseases pose significant challenges to global health and agriculture. Infections caused by invasive fungi, such as candidiasis, aspergillosis, and cryptococcosis, can be life-threatening for individuals with weakened immune systems (Perlin et al. 2015; Bongomin et al. 2017; Rodrigues and Nosanchuk 2020). Fungal infections have a major impact on global health, resulting in significant rates of illness and death (Sahu and Katwala 2022; Vitiello et al. 2023). The incidence of invasive fungal infections has risen drastically, due in part to the increasing population of immunocompromised individuals (Orlowski et al. 2017; Nanjappa and Mudalagiriyappa 2021). The limited availability of antifungal drugs and the emergence of drug resistance highlight the necessity for new and innovative antifungal treatments (Dos Reis et al. 2021; Rabaan et al. 2023). Fungi also cause significant damage to crops, food spoilage, and contamination by mycotoxins. Although current antifungal drugs are effective, they have limitations in terms of toxicity, the emergence of resistance, and environmental impact. Therefore, there is a pressing need for new antifungal options that offer improved selectivity and safety (Wu et al. 2022; Karanth et al. 2023).

Chitin, a polysaccharide made up of N-acetylglucosamine units, is a major component of fungal cell walls as well as insect and crustacean exoskeletal structures. Chitinases, or glycosyl hydrolase enzymes, break down chitin into smaller oligosaccharides or monomers, which are important for nutrient recycling, plant defense, and pathogen inhibition (Ezeilo et al. 2017; Monge et al. 2018; Jankiewicz et al. 2021). Enzymes hydrolyze β-1,4 linkages in chitin polymers, destroying fungal cell walls. Due to this ability, chitinases are considered promising tools for controlling fungal infections, especially fungicide-resistant strains (Hakeem et al. 2022; Valle-Sotelo et al. 2022). Produced by a variety of organisms, including bacteria, fungi, plants, insects, and crustaceans, chitinases serve as a defense mechanism to undermine the growth and structure of fungi by breaking down their cell wall chitin (Kumar et al. 2018).

Bacteria are known to produce chitinases with antifungal properties, which have potential applications in agriculture, medicine, and industry. Among these bacteria, B. subtilis and S. marcescens have been extensively studied for their ability to produce chitinases with antifungal activity against a range of fungal pathogens (Mamangkey et al. 2022; Dikbaş et al. 2023).

In recent years, genetic engineering techniques have provided scientists with the means to modify and optimize the properties of enzymes for specific applications. One such approach is the construction of hybrid chitinase enzymes, created by combining functional domains from different parental chitinases. The rationale behind this strategy is to create hybrid enzymes with enhanced or novel properties by utilizing the beneficial characteristics of multiple enzymes (Dubrovna et al. 2022).

Research has been conducted to investigate the ability of recombinant chitinases from different sources to combat phytopathogens and human mycoses. However, the effectiveness of individual chitinases as antifungal agents is still inferior to that of chemical fungicides, which hinders their progress in practical applications (Goodrick et al. 2001).

In this study, we aimed to engineer hybrid chitinases with heightened antifungal properties by fusing genes encoding chitinases from B. subtilis and S. marcescens. The hybrid chitinases would demonstrate enhanced antifungal activities compared to the original enzymes. Chitinase genes were cloned, fused, and expressed in E. coli. The hybrid enzymes were characterized and evaluated for their ability to inhibit fungal growth. The results provide insight into optimizing chitinases to develop novel antifungal agents.

Materials and methods

Bacterial strains isolation and identification

Two bacterial strains producing chitinase enzyme were isolated from Allium ampeloprasum plants. These plants were cultivated at BUC University in Cairo, in the School of Biotechnology.

Samples were collected from the roots and rhizospheric soil of Allium ampeloprasum plants.

The collected samples were subjected to serial dilution and plated on a nutrient agar medium. Plates were incubated at 37 °C for 24–48 h to allow bacterial growth.

Genomic DNA was extracted from bacterial strains using a commercial DNA extraction kit according to the manufacturer’s instructions. The 16S rRNA gene was then amplified by PCR using universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). The resulting PCR products were analyzed by agarose gel electrophoresis, and samples showing a distinct band at approximately 1500 bp were selected for sequencing. Sanger sequencing was performed in both the forward and reverse directions using the primers.

To identify the most similar bacterial species, the 16S rRNA gene sequences were analyzed using the BLASTN program at NCBI. ClustalW was used to align multiple sequences, including reference sequences, for further analysis. The Geneious Prime 2021.1.1 software was used to construct a phylogenetic tree using the Maximum Likelihood method. The Kimura 2-parameter method was employed to compute evolutionary distances.

Chitinase gene selection and cloning

The chitinase gene Chi-Gh18-FN3_CBD was selected for cloning based on the reported antifungal activities of its component domains (Kitajima et al. 2012; Chen et al. 2023; Wang et al. 2023a). This hybrid chitinase gene consists of the signal peptide and catalytic domain from S. marcescens chitinase A (SignalP-TM, ChitinaseA_N, and Glyco_hydro_18) (Fig. 1a) fused to the fibronectin type III (FN3) and carbohydrate-binding (CBD) domains from B. subtilis (Fig. 1b). This hybrid chitinase gene consists of the signal peptide and catalytic domain shown in Fig. 1c.

Fig. 1
figure 1

Structure of (a) S. marcescens, (b) B. subtilis, and (c) the hybrid chitinase gene with its domains

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The S. marcescens chitinase A gene was amplified from genomic DNA using PCR primers designed based on the reported sequence (GenBank accession AF069131.1). The FN3 and CBD domains were amplified from B. subtilis genomic DNA using primers designed based on the reported sequence (GenBank accession GQ855217.1). Supplementary material Table S1 shows the primer design performed using Snapgene 6.1 software. The PCR products were gel purified, digested with appropriate restriction enzymes, and ligated together to form the hybrid Chi-Gh18-FN3_CBD construct. This construct was sequence verified before subcloning into the protein expression vector pET-28a ( +) (Fig. S1).

The B. subtilis chitinase gene was amplified from genomic DNA by PCR using primers designed to add XhoI and BamHI restriction sites. The PCR product and pET-28a vector were digested with XhoI and BamHI, ligated together with T4 DNA ligase, and transformed into E. coli BL21(DE3) cells (Fig. S2). Transformants were selected on LB-kanamycin agar plates.

The S. marcescens chitinase gene was amplified from genomic DNA by PCR using primers designed to add NcoI and XhoI restriction sites. The PCR product and pET-28a vector were digested with NcoI and XhoI, ligated together with T4 DNA ligase, and transformed into E. coli BL21(DE3) cells (Fig. S3). Transformants were selected on LB-kanamycin agar plates.

The hybrid chitinase gene was constructed from two fragments a 1350 bp fragment encoding the S. marcescens chitinase and a 450 bp fragment encoding the B. subtilis chitinase. The fragments were amplified by PCR using specific primers designed to add XhoI/BamHI sites to the S. marcescens fragment and NcoI/BamHI sites to the B. subtilis fragment. The PCR products were digested with the appropriate enzymes and ligated together using T4 DNA ligase. The ligated hybrid gene was then cloned into the pET-28a ( +) expression vector and transformed into E. coli BL21(DE3) cells (Fig. S4). Transformants were selected on LB-kanamycin agar plates.

The plasmid constructs were transformed into E. coli DH5α competent cells and plated on LB agar with appropriate antibiotics for selection. Single colonies were inoculated into LB media, grown overnight, and plasmid DNA was isolated using the QIAprep Spin Miniprep Kit. The isolated plasmids were digested with restriction enzymes and run on 1% agarose gels. Bands containing the inserts were excised and DNA was purified with the QIAquick Gel Extraction Kit.

Recombinant protein expression and purification

For chitinase expression, E. coli DH5α clones were inoculated into 50 ml BMGY medium (1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 100 mM potassium phosphate pH 6.0, 1% glycerol) in 250 ml baffled flasks. Cultures were incubated at 37 °C and 250 rpm until OD600 of 0.2–0.6 was reached. The validated plasmid constructs were transformed into E. coli BL21 cells for protein expression. Protein expression was induced with IPTG.

The cultures were centrifuged at 8000 × g for 10 min and the supernatant containing secreted chitinases purified by immobilized metal ion affinity chromatography on Ni–NTA agarose resin (Qiagen). The supernatant was mixed with Ni–NTA resin pre-equilibrated with binding buffer (50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0) and incubated overnight at 4 °C with gentle agitation. The protein-resin mixture was loaded onto a column and washed with 10 column volumes of binding buffer followed by 6 column volumes of wash buffer (50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, pH 8.0). The chitinases were eluted with elution buffer (50 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole, pH 8.0) and collected in 1 ml fractions.

Protein gel electrophoresis and in silico protein analysis

The molecular weights and isoelectric points of three chitinase proteins were analyzed using protein gel electrophoresis and CLC Workbench software 5.5. The eluted protein fractions included B. subtilis chitinase (Bs-chi), S. marcescens chitinase (Sm-chi), and a hybrid chitinase (H-chi) were analyzed by 12% SDS-PAGE and those containing chitinase were pooled and dialyzed against 20 mM Tris–HCl pH 8 overnight at 4 °C to remove imidazole. The purified proteins were concentrated using Amicon Ultra centrifugal filters (10 kDa MWCO), filter sterilized, and protein concentration determined by Bradford assay using bovine serum albumin as standard. Electrophoresis was conducted at 100V for 2 h. The gel was stained with Coomassie blue to visualize the protein bands, and the molecular weights of the proteins were estimated by comparison to protein standards run on the same gel.

The Thermo Scientific™ PageRuler™ Plus Prestained Protein Ladder, with molecular weights spanning from 10 to 250 kDa, was utilized. The theoretical isoelectric points were determined using the Compute pI/Mw tool in CLC Workbench using the protein sequences.

Chitinase activity assays

The fluorogenic substrate 4-methylumbelliferyl N-acetyl-β-D-glucosaminide (4-MUG) (Sigma) was used to measure chitinase activity. Purified enzymes (10 µl) were incubated with 100 µM 4-MUG in 50 mM citrate phosphate buffer pH 6.5 at 37 °C. The fluorescence of liberated 4-methyl-umbelliferone was measured at excitation and emission wavelengths of 365 and 450 nm, respectively, using a Cytation 5 multi-mode plate reader (BioTek). A standard curve of 4-methyl-umbelliferone was used to calculate activity. One unit of chitinase activity was defined as the amount of enzyme required to produce 1 µmol of 4-methyl-umbelliferone per minute at 37 °C.

Antifungal activity testing

An agar well diffusion assay was performed to assess the antifungal properties of the purified recombinant chitinases against four fungal species: Candida albicans ATCC 10231, Alternaria solani ATCC 32904, F. oxysporum MTCC 284, and Rhizoctonia solani AG 1 (ATCC 58946). Each chitinase (Bs-chi, Sm-chi, and H-chi) was introduced into wells on plates that had been previously inoculated with fungal plugs. For inoculation, a 5 mm fungal plug was placed in the center of each PDA plate, and 5 mm wells were created using a sterile cork borer. Following incubation, the zones of inhibition were measured.

A volume of 10 µl of the prepared chitinase solution was then added to each well. The plates were incubated at 28 °C for C. albicans ATCC 10231 and at 25 °C for A. solani ATCC 32904, F. oxysporum MTCC 284, and R. solani AG 1 (ATCC 58946). After 48 h, the zones of inhibition were measured. The minimum inhibitory concentration (MIC) of C. albicans was assessed using a microbroth dilution assay. Chitinases were serially diluted twofold in 96-well plates containing potato dextrose broth inoculated with 105 cells/ml. Each treatment was performed in triplicate to ensure statistical accuracy and reproducibility. The plates were then incubated at the specified temperatures for 24–48 h and visually inspected for the absence of growth to determine the MIC.

3D homology models of chitinases

The amino acid sequences of chitinases from B. subtills and S. marcescens were acquired from the UniProt database (accession ID AAC23715.1 and ACX42071.1, respectively). A search was conducted in the Protein Data Bank (PDB) to find suitable template structures that shared at least 30% sequence identity. The template sequences identified for B. subtills were 1ITX_A, 6BT9_B, 6KST_A, 6KXM_A, 5GZT_B, and 5GZU, while the template sequences for S. marcescens chitinases were 1FFQ_A, 1NH6_A, 1CTN_A, 5Z7M_A, and 5ZL9_A. From these, the hybrid protein selected 1CTN_A, 5Z7M_A, 5ZL9_A, 1EDQ_A, and 1RD6_A-1 based on the highest sequence identity, coverage, and resolution. The sequence alignment between the target and template was generated using the Prime module in the Schrodinger suite (Schrödinger 2019a, 2018, 2022, 2019b; Sheikh et al. 2019).

In silico docking activity

Schrödinger software was used to build the 3D structures of the chitinases from B. subtilis, S. marcescens, and the hybrid protein. Molecular docking was performed using the Glide module in the Schrödinger suite. The preparation of protein and ligand structures was conducted using the Protein Preparation Wizard and LigPrep modules. Grid generation was focused on the active site residues of the chitinases. Flexible docking was executed with standard precision (SP) scoring. The best docking poses were analyzed to identify the interactions using Maestro (Schrödinger 2015; Schrödinger 2020).

Results

Bacterial identification

The identification of the bacterial isolates B. subtills and S. marcescens was performed using 16S rRNA gene sequencing. Genomic DNA extraction and PCR amplification with universal 16S primers resulted in PCR products of 976 bp and 925 bp, respectively. Sanger sequencing and BLAST analysis revealed a 98.3% similarity to B. subtills strain BAD-7067 (Acc NO. MF319828) for the B. subtills isolate (Fig. 2a). Phylogenetic analysis of the S. marcescens isolate showed a 98.8% similarity to S. marcescens strain Gh-Fa-6 (Acc NO. OR251114) and S. marcescens strain Gh-Fa-5 (Acc NO. OR251113) (Fig. 2b). Sequence data for the chitinase gene used in this study have been deposited in the GenBank database under the submission number SUB14029460.

Fig. 2
figure 2

Identification and phylogenetic analysis of (a) B. subtills and (b) S. marcescens isolates using 16S rRNA gene sequencing

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Cloning and sequence analysis

The chitinase gene from S. marcescens (Sm-chi) and B. subtills (Bs-chi) were amplified by PCR using gene-specific primers, and also the hybrid chitinase protein fragment (Supplementary material Table S2). The PCR product of B. subtills (Bs-chi)(Bs-Chi), S. marcescens (Sm-chi), and the two fragments of hybrid chitinase showed product length of 1809, 1710, and 1350 bp fragment encoding the S. marcescens chitinase part and a 450 bp fragment encoding the B. subtilis chitinase the hybrid chitinase, respectively (Fig. S5). The chitinase gene and PCR fragment were ligated into the pET-28a( +) expression vector and transformed into E. coli DH5α. Colony PCR screening identified positive recombinant clones. The results of the gel electrophoresis showed that the hybrid chitinase plasmid of 7007 bp, the chitinase plasmid of B. subtilis of 7126 bp, and the chitinase-containing plasmid from S. marcescens of 6929 bp were all successfully cloned into the PET-28a( +) expression vector.

Production and purification of chitinase proteins

The chitinase genes from S. marcescens (Sm-chi), B. subtilis (Bs-chi), and the hybrid PCR product were inserted into the pET-28a( +) plasmid and transformed into E. coli BL21(DE3) cells. The expression of the chitinases was induced with IPTG, and the (His) 6-tagged chitinase proteins from S. marcescens (Sm-Chi), B. subtilis (Bs-Chi), and the hybrid were purified using Ni–NTA affinity chromatography. Nickel column purification resulted in 5–10 mg of purified protein per liter of bacterial culture. Desalting removed imidazole and other contaminants, ensuring pure proteins in PBS for downstream applications. Analysis using CLC Workbench software v5.5 confirmed the theoretical molecular weights of the protein sequences, and SDS-PAGE showed high expression levels of all three chitinases in the E. coli cells. Each chitinase had a distinct band on the gel with molecular weights of 65.894 kDa for Bs-chi, 61.081 kDa for Sm-chiA, and 63.429 kDa for H-chi (Fig. 3 and Supplementary material Table S2). The computed isoelectric points were 5.27 for Bs-chi, 6.56 for Sm-chi, and 6.25 for H-chi.

Fig. 3
figure 3

SDS-PAGE analysis of purified chitinase proteins with (His) 6-Tag expressed in E. coli BL21(DE3)

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Enzyme characterization

Table 1 displays the activity of the purified recombinant chitinases, which was assessed using the fluorogenic substrate 4-MUG. The hybrid chitinase exhibited the highest activity at 120 U/mg, while Sm-chi had an activity of 95 U/mg and Bs-chi had an activity of 65 U/mg, all under standard assay conditions of pH 6.5 and 37 °C.

Table 1 Chitinase activities of purified enzymes measured using 4-MUG substrate
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The kinetic parameters of the purified recombinant chitinases H-chi hybrid chitinase, Sm-chi chitinase, and Bs-chi chitinase were determined using the fluorogenic substrate 4-MUG (Table 2). The hybrid chitinase (H-chi) displayed the highest Vmax of 389 µmol/min/mg and catalytic efficiency (kcat/Km) of 4.3 × 106 M-1s-1 among the three chitinases.

Table 2 Kinetic parameters of the chitinases
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Antifungal activity

The antifungal efficacy of three chitinase enzymes Bs-chi, Sm-chi, and Hybrid chitinase (H-chi) was tested against four fungal species: C. albicans, A. solani, F. oxysporum, and R. solani AG 1 (ATCC 58946). Bs-chi demonstrated inhibition values of 12 ± 1.2 mm for C. albicans, 10 ± 0.9 mm for A. solani, 7 ± 0.8 mm for F. oxysporum, and 14 ± 1.3 mm for R. solani AG 1 (ATCC 58946). Similarly, Sm-chi showed inhibition values of 12 ± 1.1 mm, 11 ± 1.0 mm, 7 ± 0.7 mm, and 16 ± 1.4 mm against these respective fungi. H-chi exhibited the highest antifungal activity, with inhibition values of 22 ± 1.5 mm for C. albicans, 18 ± 1.3 mm for A. solani, 16 ± 1.2 mm for F. oxysporum, and 20 ± 1.5 mm for R. solani (Table 3 and Fig. 4).

Table 3 Antifungal activity of chitinase enzymes against C. albicans ATCC 10231, A. solaniATCC 32904, F. oxysporum MTCC 284, and R. solani AG 1 (ATCC 58946) (mean ± SD)
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Fig. 4
figure 4

Agar diffusion assay of purified recombinant Chitinases against C. albicans ATCC 10231, A. solani ATCC 32904, F. oxysporum MTCC 284, and R. solani AG 1 (ATCC 58946)

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The antifungal activities of various agents against C. albicans ATCC 10231 were assessed, revealing distinct efficacy profiles. Fluconazole exhibited MIC values ranging from 2.064 to 120 µg/ml, indicating its potency at lower concentrations but reduced efficacy at higher doses. Itraconazole demonstrated better minimum MIC values (1.072 µg/ml) and was effective up to 58 µg/ml, suggesting a more consistent antifungal profile. Voriconazole displayed moderate activity with MIC values between 2.082 and 65 µg/ml, while Amphotericin B showed potency with minimum and maximum MIC values of 3.251 and 75 µg/mL, respectively. Among the chitinase enzymes, Bs-chi and Sm-chi had similar MIC ranges (2.893–100 U/ml and 2.904–90 U/ml, respectively), indicating moderate antifungal properties. Notably, the Hybrid chitinase (H-chi) exhibited significantly lower MIC values, ranging from 1.224 to 30 U/ml, suggesting enhanced antifungal efficacy likely due to synergistic effects and improved enzymatic activity. Thus, H-chi stands out as a particularly effective antifungal agent against C. albicans ATCC 10231 compared to standard antifungals and other chitinases tested (Table 4).

Table 4 Antifungal activity against C. albicans ATCC 10231 and MIC values
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3D homology models of chitinases

The construction of a homology model based on the alignment of the target and template was carried out using the Prime software. The initial model was enhanced by optimizing the hydrogen bond network and reducing clashes between side chains. Further refinement was achieved through several iterations of Prime energy minimization and elimination of unfavorable side chain conformations.

The B. subtilis chitinase (519 aa) was modeled using the chitinase structure from B. circulans (PDB ID: 1ED7) as template showing 64% sequence identity. The S. marcescens chitinase (577 aa) was modeled using S. marcescens chitinase A structure (PDB ID: 1CTN) sharing 99% sequence identity. For the hybrid chitinase (820 aa), the B. circulans chitinase structure and S. marcescens chitinase A structure were used as templates for the respective domains.

The final models showed > 90% residues in the favored regions of Ramachandran plot. The DOPE scores were − 42678.59769, − 48121.39062, and − 57327.61719 for B. subtilis, S. marcescens, and hybrid chitinase models, respectively. GA341 scores were 1.0 for all the models (Fig. 5).

Fig. 5
figure 5

3D model of (a) B. subtilis chitinase, (b) S. marcescens chitinase, and (c) hybrid chitinase

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Molecular modeling

Molecular docking predicted the hybrid chitinase binds chitin through residues from both B. subtilis (Bs-chi) and S. marcescens (Sm-chi) domains (Fig. 6). Several hydrogen bonds and hydrophobic interactions mediate binding in the fused bi-domain structure. The model suggests improved substrate binding enables more efficient catalytic cleavage by the hybrid enzyme.

Fig. 6
figure 6

Molecular docking of (a) B. subtilis chitinase, (b) S. marcescens chitinase, and (c) hybrid chitinase with chitin

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The docking scores and interacting residues of the chitinases with chitin are summarized in Table 5. The hybrid chitinase showed the lowest (most negative) docking score of − 7.859 kcal/mol compared to B. subtilis (− 4.532 kcal/mol) and S. marcescens (− 5.785 kcal/mol) chitinases.

Table 5 Molecular docking results of purified recombinant chitinases with chitin
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The hybrid chitinase formed 9 hydrogen bonds with chitin involving residues Trp275, Thr276, Glu315, Asp391, Gln404, Leu455, Ala517, and Asp518. In contrast, B. subtilis and S. marcescens chitinases formed only 4 and 3 H-bonds, respectively (Fig. 6).

Enzyme activity and kinetics

Effect of enzyme concentration on chitinase activity

The chitinase activity for various enzyme concentrations (µg/µl) of B. subtilis, S. marcescens, and the hybrid enzyme is illustrated in Fig. 7. The activity of the chitinases from B. subtilis, S. marcescens, and the hybrid enzyme increased steadily with higher enzyme concentrations tested. The hybrid chitinase demonstrated more significant activity enhancements compared to the native enzymes at multiple concentrations. The hybrid chitinase enzyme demonstrated more than a twofold increase in activity (30.46 U/ml) at a concentration of 25 μg/ml, surpassing that of B. subtilis (12.81 U/ml). The most notable enhancement was observed at 150 µg/ml, with the hybrid enzyme showing an activity of 83.69 U/ml, outperforming the activities of both B. subtilis (65.45 U/ml) and S. marcescens (63.05 U/ml). While the activity of the native enzymes plateaued after 175 µg/ml, indicating saturation of their catalytic capacity, the activity of the hybrid chitinase continued to increase, reaching 88.47 U/ml at the maximum concentration of 200 µg/ml. The study analyzed the Michaelis–Menten kinetics of chitinase activity in three distinct strains and established the best-fit values for Vmax and Km. The findings indicated that the hybrid strain exhibited the highest Vmax value at 145.3, surpassing those of B. subtilis (128.8) and S. marcescens (133.6), respectively. Conversely, the hybrid strain displayed the lowest Km value at 125.7 compared to B. subtilis (148.2) and S. marcescens (169.0) strains as detailed in Table 6.

Fig. 7
figure 7

Chitinase activity of B. subtilis, S. marcescens, and hybrid chitinase  at (a) different enzyme concentrations (µM),(b) different time intervals, (c) different pH values, and (d) different temperatures. Bs-chi (B. subtilis chitinase), Sm-chi (S. marcescens chitinase), and H-chi (hybrid chitinase)

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Table 6 Michaelis–Menten kinetic for enzyme concentration on the activity of chitinase
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Effect of reaction incubation time on chitinase activity

The initial reaction velocity of all three chitinases increased rapidly within the first 30 min of incubation. The hybrid chitinase consistently exhibited the highest activity across all time points tested. Its activity reached a peak of 75.44 U/ml after 40 min, which was approximately 1.3 times higher than the peak activity of 46.13 U/ml observed for S. marcescens chitinase. Between 40 and 70 min, the enzyme activity plateaued and started to gradually decrease, suggesting a possible restriction in substrate availability. The decrease was most noticeable for B. subtilis chitinase, dropping from 61.69 to 51.67 U/ml. In contrast, hybrid chitinase maintained a higher residual activity of 66.67 U/ml by the 70-min (Fig. 7a). To analyze the kinetic properties of chitinase activity over time for B. subtilis, S. marcescens, and hybrid strains, Michaelis–Menten kinetic analysis was conducted. Table 7 presents the best-fit values of Vmax and Km, along with their corresponding 95% confidence intervals (CIs).

Table 7 Michaelis–Menten kinetic for incubation time on the activity of chitinase
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Effect of temperature on enzyme activity

The temperature range tested revealed optimal activity for all three chitinases. The chitinase from B. subtilis showed the highest activity at 37 °C (31.6 U/ml), while S. marcescens exhibited peak activity at 30 °C (27.6 U/ml). The hybrid chitinase displayed optimal activity between 30 and 37 °C (26.3–33.2 U/mL). As temperatures exceeded 40 °C, enzyme activity decreased, likely due to enzyme structure instability and unfolding. B. subtilis experienced the most significant decline, dropping by 34% at 45 °C. In contrast, the hybrid maintained a higher residual activity at 28.8 U/mg, equivalent to 86% of its maximum activity (Fig. 7b).

Effect of pH on enzyme activity

Figure 7c illustrates the chitinase enzyme activity (in U/ml) of B. subtilis, S. marcescens, and their hybrid variant across varying pH levels. The assessment covered pH levels from 5 to 8. The findings reveal that the hybrid variant exhibited higher chitinase enzyme activity compared to the individual strains across most pH conditions. Specifically, at pH 5, B. subtilis displayed a chitinase activity of 21.14 U/ml, while S. marcescens showed 18.03 U/ml. The chitinase activity of the hybrid variant was recorded at 20.10 U/ml, positioning it between the values of the two separate strains. As the pH levels increased, the chitinase activity of all three strains also increased. It was especially noticeable that the hybrid strain exhibited higher chitinase activity compared to the individual strains, with a significant contrast noted at pH 7.5 (Fig. 7b).

Discussion

Chitinases, enzymes that catalyze the degradation of chitin, an essential component of fungal cell walls, have shown significant promise in antifungal applications. The ability of these enzymes to break down chitin provides a unique mechanism for combating fungal infections, making them a potential basis for the development of novel antifungal therapies (Monge et al. 2018; Pentekhina et al. 2020; Tran et al. 2022). Chitinases exhibit antifungal properties by specifically targeting the chitin present in fungal cell walls, leading to cell wall degradation and subsequent fungal cell death. This mechanism is advantageous as it directly disrupts the structural integrity of fungal cells.

Based on their effectiveness in halting the growth of harmful fungi, these enzymes could be a good starting point for investigating new antifungal therapies (De Medeiros et al. 2018; Cd et al. 2021; Dikbaş et al. 2023). The use of chitinase enzymes, which degrade chitin in fungal cell walls, is a promising strategy. B. subtilis and S. marcescens produce enzymes that have antifungal properties. Some bacteria, like B. subtilis and S. marcescens, have antifungal characteristics due to the chitinases they produce.

The effectiveness of fungal inhibition can be increased by combining functional domains from different bacterial chitinases to form hybrid enzymes (Oranusi and Trinci 1985; Senol et al. 2014).

The aim of this research was to create genetically modified hybrid chitinase enzymes with improved antifungal properties by merging functional domains from the chitinases of B. subtilis and S. marcescens. To achieve this, chitinase genes from both bacterial species were cloned and then fused using overlap extension PCR. These hybrid constructs were subsequently expressed in E. coli, enabling the production of recombinant hybrid chitinases.

Identification of bacterial isolates is crucial as it provides insights into their origin, potential pathogenicity, and unique characteristics. This understanding enhances our knowledge of microbial interactions with their environments. Additionally, accurately identifying bacterial strains is essential for designing and interpreting future experiments involving these isolates. In this study, precise identification of bacterial isolates was paramount to understanding their biological properties, potential pathogenic abilities, and ecological significance. The isolates were identified using 16S rRNA gene sequencing, a method known for its conserved nature and inclusion of variable and conserved regions (Iskandar et al. 2021; Ahmed et al. 2022). This sequencing technique effectively identified the bacterial isolates at the species level. After identification, these isolates were used to produce and purify chitinases, enzymes that have potential applications in agriculture and biotechnology. Successful identification and cloning of chitinase genes allowed for the expression and testing of enzyme activity (Nguyen Hoang et al. 2022). BLAST analysis of the B. subtilis isolate showed a 98.3% similarity to the B. subtilis strain BAD-7067, suggesting that the isolate is very likely the same species as the reference strain. This supports identifying the isolate as B. subtilis. Similarly, phylogenetic analysis of the S. marcescens isolate revealed a 98.8% similarity to S. marcescens strains Gh-Fa-6 and Gh-Fa-5, indicating that the isolate is likely a strain of S. marcescens. BLAST analysis of the B. subtilis isolate revealed a 98.3% similarity to the B. subtilis strain BAD-7067, indicating that the isolate is very likely the same species as the reference strain. This supports the identification of the isolate as B. subtilis. Similarly, phylogenetic analysis of the S. marcescens isolate showed a 98.8% similarity to S. marcescens strains Gh-Fa-6 and Gh-Fa-5, suggesting that the isolate is likely a strain of S. marcescens.

In this study, a novel hybrid chitinase gene called Chi-Gh18-FN3_CBD (H-chi) was generated. To construct this gene, the signal peptide and catalytic domain from S. marcescens chitinase A were combined with the FN3 and CBD domains from B. subtilis. This chitinase gene was specifically chosen because previous studies have shown that the individual domains possess antifungal properties (Jones et al. 1986; Suzuki et al. 1999; Van Aalten et al. 2000; Chen et al. 2004; Sha et al. 2016). Our objective was to enhance the performance of the chitinase enzyme by creating a hybrid gene. Chitinase enzymes with high efficiency and effectiveness are essential for various applications, such as degradation of chitin-based waste, management of fungal diseases, and production of low molecular weight chitin oligomers. The hybrid chitinase gene Chi-Gh18-FN3_CBD would exhibit improved efficacy due to the collaborative action of its diverse components.

The hybrid genes of chitinase were produced by amplifying the S. marcescens chitinase A gene and the FN3/CBD domains from B. subtilis genomic DNA using PCR. The resulting PCR products were purified and then digested with specific restriction enzymes. These fragments were then ligated together to create the hybrid Chi-Gh18-FN3_CBD construct. The hybrid construct was further inserted into the protein expression vector pET-28a ( +) and transformed into E. coli BL21(DE3) cells for protein production and purification. The successful construction of the hybrid gene and its expression in E. coli BL21(DE3) cells were confirmed through DNA sequencing and western blot analysis.

The development of a hybrid chitinase gene presents a promising strategy for enhancing the efficacy of chitinase in diverse applications. By incorporating distinct domains from various sources, including the signal peptide, catalytic domain, and FN3/CBD domains, the objective was to generate a chitinase enzyme with enhanced specificity for substrates, improved binding affinity, and more efficient degradation capabilities. Furthermore, the utilization of the pET-28a ( +) expression vector was pivotal in this study as it facilitated the production of substantial quantities of the recombinant hybrid chitinase protein.

In this study, the chitinase genes from S. marcescens (Sm-chi) and B. subtilis (Bs-chi) were amplified using gene-specific primers, along with a fragment encoding the hybrid chitinase protein. The PCR products of Bs-chi, Sm-chi, and the hybrid chitinase fragment were of 1809, 1710, and 1350 bp in length, respectively. These fragments were then ligated into the pET-28a( +) expression vector and transformed into E. coli DH5α cells.

Colony PCR screening was performed to identify positive recombinant clones. The results of gel electrophoresis confirmed the successful insertion of the chitinase genes and the hybrid chitinase fragment into the pET-28a( +) vector. The sizes of the plasmids containing the hybrid chitinase, B. subtilis chitinase, and S. marcescens chitinase were determined to be 7007, 7126, and 6929 bp, respectively.

These cloning and sequence analysis results demonstrate the successful construction of chitinase-expressing plasmids and the presence of the desired gene sequences in the transformed E. coli DH5α cells. The use of gene-specific primers and the appropriate restriction sites in the pET-28a( +) vector allowed for efficient cloning of the chitinase genes.

The pET-28a( +) vector is a commonly used expression vector that enables high-level protein expression in E. coli (Ghavim et al. 2017). The inclusion of a His-tag in the vector facilitates the purification of the expressed proteins using immobilized metal ion affinity chromatography, as described earlier in the methods section (Wanarska et al. 2005; Ghavim et al. 2017).

The theoretical molecular weights of the chitinase proteins were confirmed using CLC Workbench v5.5, based on their protein sequences. Additionally, SDS-PAGE analysis was performed to evaluate the expression levels of the chitinases in E. coli cells. The gel showed clear bands that corresponded to each chitinase, indicating successful protein expression. The molecular weights of the chitinases were estimated as 65.894 kDa for Bs-chi, 61.081 kDa for Sm-chi, and 63.429 kDa for the hybrid chitinase (H-chi). In addition, the isoelectric points (pIs) of the chitinase proteins were calculated as 5.27 for Bs-hi, 6.56 for Sm-chi, and 6.25 for H-chi. These pI values offer valuable information about the proteins’ charge properties and can be beneficial for future investigations concerning their stability, interactions, and potential uses. The cloning strategy and expression system employed in this study were proven to be effective as evidenced by the successful production and purification of the chitinase proteins.

Under the assay conditions of pH 6.5 and 37 °C, the H-chi hybrid chitinase demonstrated the greatest activity at 120 U/mg, followed by the Sm-chi chitinase at 95 U/mg, and the Bs-chi chitinase at 65 U/mg. These findings suggest that the hybrid chitinase exhibited the most efficient catalytic performance in breaking down the 4-MUG substrate.

The hybrid chitinase (H-chi) demonstrated the lowest Km value, indicating a greater affinity for the 4-MUG substrate compared to the other two chitinases. This suggests that H-chi is more efficient in degrading chitin. H-chi also had the highest Vmax value, representing the maximum velocity of the enzyme-catalyzed reaction, at 389 µmol/min/mg. In addition, H-chi had the highest kcat value at 650 s−1, indicating that it converts a greater number of substrate molecules into product per second at each active site. The kcat/Km ratio, which reflects the catalytic efficiency of the enzyme, was also highest for H-chi at 4.3 × 106 M−1s−1.

In terms of kinetic parameters, H-chi demonstrated the highest catalytic efficiency and substrate affinity compared to the other two chitinases. Sm-chi had lower values for Km, Vmax, kcat, and kcat/Km when compared to H-chi. Bs-chi exhibited the lowest values for all kinetic parameters, suggesting relatively lower catalytic efficiency and substrate affinity. Many studies showed that examines how terminal modifications affect various enzymes. Truncating the C-terminal end of the chitinase from Bacillus licheniformis resulted in increased thermostability without notably changing substrate binding and hydrolysis (Chuang et al. 2008). In the case of alkaline α-amylase, combining C-terminal truncation with N-terminal oligopeptide fusion led to substantial improvements in specific activity and catalytic efficiency (Yang et al. 2013). For ruminal xylanases, a proline-rich sequence at the C-terminal end enhanced catalytic efficiency, expanded the optimal temperature and pH ranges, and improved xylose release (Li et al. 2014). Conversely, for a GH10 xylanase from Volvariella volvacea, truncations at both the N- and C-terminal ends increased activity and thermostability but reduced SDS resistance (Zheng et al. 2016). These studies illustrate that terminal modifications can significantly influence enzyme properties, such as activity, stability, and substrate specificity, offering valuable insights for protein engineering strategies aimed at enhancing enzyme performance for various applications.

The results indicate that the hybrid chitinase (H-hi) protein outperforms the other two chitinases in terms of effectiveness and activity. This knowledge is crucial for future research exploring the potential uses of these chitinases in diverse sectors, including agriculture, waste management, and biofuel manufacturing.

The antifungal activity of hybrid chitinase (H-chi) exhibits significantly greater efficacy compared to Bs-chi and Sm-chi across all tested fungal species. Remarkably, the inhibition values of H-Chi against C. albicans (22 ± 1.5 mm), A. solani (18 ± 1.3 mm), and F. oxysporum (16 ± 1.2 mm) were nearly double those of the non-hybrid chitinases, implying a superior synergistic or enhanced enzymatic mechanism behind its improved performance. This increased activity of H-chi underscores its potential as a potent antifungal agent, likely due to the combined attributes of chitinases from different sources. The consistent effectiveness of H-chi across multiple fungi also indicates a broader spectrum of applicability, making it a promising candidate for future antifungal applications and research.

The antifungal activity of various agents against C. albicans highlights distinct efficacy profiles, providing valuable insights for potential therapeutic applications. Among the standard antifungal agents tested, Fluconazole showed a relatively wide range of MIC values (2.064 to 120 µg/mL), which underscores its potent antifungal action at lower concentrations but also suggests reduced effectiveness at higher doses. This variability may necessitate careful dose adjustment to maximize therapeutic benefit while minimizing potential resistance or side effects. Itraconazole demonstrated more consistent antifungal activity with MIC values of 1.072 to 58 µg/mL, implying its suitability as a reliable antifungal agent for treating C. albicans infections. Voriconazole’s MIC values (2.082 to 65 µg/mL) indicate moderate efficacy, positioning it as an alternative in situations where other antifungals are less effective. Amphotericin B, known for its broad-spectrum antifungal activity, exhibited MIC values ranging from 3.251 to 75 µg/mL. Its higher minimum MIC underscores the need for precise dosing to balance efficacy and toxicity. The introduction of chitinase enzymes offers a novel approach to antifungal therapy. Bs-chi and Sm-chi yielded comparable MIC ranges (2.893–100 U/mL and 2.904–90 U/mL, respectively). These enzymes’ moderate antifungal properties suggest their potential as adjunctive treatments, possibly enhancing the activity of traditional antifungals or providing alternative mechanisms of action. Of particular interest is the hybrid chitinase (H-chi), which exhibited significantly lower MIC values (1.224 to 30 µg/mL), indicating superior antifungal efficacy. The enhanced performance of H-chi may be attributed to synergistic effects that arise from the hybridization of chitinase enzymes, resulting in improved enzymatic activity and broader targeting of fungal cell walls. This makes H-chi a promising candidate for further development and clinical testing, potentially offering a more potent and targeted antifungal therapy for C. albicans infections.

In order to understand the structural basis of the antifungal activity of the chitinases, 3D models for each chitinase were constructed using the Prime software. These models were then refined through several iterations of energy minimization and removal of unfavorable side chain conformations. The chitinases from B. subtilis and S. marcescens were modeled based on their respective template structures, which had high sequence identity. The hybrid chitinase model included relevant domains from both template structures. The final models exhibited favorable characteristics, with over 90% of residues falling within the favored regions of the Ramachandran plot. Additionally, the DOPE scores and GA341 scores indicated the overall quality and reliability of the models. The successful modeling of the chitinases provides a structural basis for understanding their antifungal activity. By comparing the models, we can discern potential structural differences that contribute to the superior antifungal properties of hybrid chitinase. These insights can guide further optimization and engineering of chitinases for enhanced antifungal activity.

The molecular docking analysis provides insights into the binding interaction between the chitinases and the chitin substrate. The hybrid chitinase showed improved binding affinity and more favorable docking score compared to the individual B. subtilis and S. marcescens chitinases. This can be attributed to contributions from functional residues of both domains of the hybrid enzyme (Sasaki et al. 2003; Saadhali et al. 2016; Jain et al. 2021).

The results indicated that the hybrid chitinase had the highest affinity for chitin, as shown by its most negative docking score of − 7.859 kcal/mol, compared to docking scores of − 4.532 kcal/mol for Bs-chi and − 5.785 kcal/mol for Sm-chi chitinases. The docking model revealed that the hybrid chitinase binds to chitin using residues from both Bs-chi and Sm-chi domains. Specifically, the hybrid chitinase formed 9 hydrogen bonds with chitin involving residues Trp275, Thr276, Glu315, Asp391, Gln404, Leu455, Ala517, and Asp518, while B. subtilis and S. marcescens chitinases formed only 4 and 3 hydrogen bonds, respectively. The increased number of hydrogen bonds likely contributes to the stronger binding and lower docking score of the hybrid enzyme. This expanded hydrogen bonding network involves conserved catalytic residues from both domains, including Asp391 from the Sm-chi domain and Asp518 from the Bs-chi domain. Furthermore, the binding cleft contains several hydrophobic residues, such as Trp275, Thr276, Leu455, and Ala517, which engage in hydrophobic interactions to enhance the stability of chitin binding. These interactions, along with polar interactions, likely contribute to the proper alignment of the substrate, facilitating efficient catalytic cleavage.

The superior performance of the hybrid chitinase even at high enzyme levels implies an enhanced enzymatic efficiency compared to the native chitinases. Likely explanations include a higher substrate turnover rate, lower Km permitting greater saturation, and improved stability at high concentrations mitigating denaturation. According to the Michaelis–Menten analysis, the hybrid strain has a higher Vmax value and a lower Km value compared to the other two strains (Zakarlassen et al. 2009; Ooi et al. 2021).

The high Vmax value in the hybrid strain suggests that it has a high capacity for chitin degradation as it can hydrolyze large amounts of chitin in a given time. This could be attributed to either higher enzyme expression or higher stability of the chitinase enzyme in the hybrid strain compared to the other two strains. On the other hand, the low Km value in the hybrid strain indicates a higher affinity of the chitinase enzyme towards chitin and a lower substrate concentration required to achieve half of its maximum activity. This indicates that the chitinase enzyme from the hybrid strain is more efficient in chitin degradation compared to the other two strains (El-Sayed et al. 2017; Pawaskar et al. 2021; Wang et al. 2023b).

The significant increase in the initial enzyme activity suggests that these enzymes probably have rapid turnover rates, leading to quick catalysis at the beginning. The decline observed during extended periods of incubation might be attributed to either product inhibition or enzyme instability (Goryanova et al. 2015).

Our results indicate that all three strains displayed Michaelis–Menten kinetics over time. The determined Vmax and Km values represent the maximum enzymatic activity and the substrate concentration needed for half-maximal activity, respectively. The Vmax values for B. subtilis, S. marcescens, and the hybrid enzyme were 78.76, 94.95, and 99.84, respectively, suggesting that the Hybrid strain exhibited the highest catalytic activity, followed by S. marcescens and B. subtilis. The Km values for B. subtilis, S. marcescens, and the hybrid enzyme were 20.56, 53.35, and 21.30, respectively, indicating that the hybrid enzyme displayed the greatest affinity for the chitin substrate, followed by B. subtilis and S. marcescens. The 95% confidence intervals reveal that the Vmax and Km values fall within a range, reflecting the variation in kinetic parameters among the cell population.

The moderate thermophilicity of these chitinases makes them suitable biocatalysts for industrial processes often conducted at 30–40 °C. The hybrid enzyme seems to tolerate higher temperatures above its optimum better than the native counterparts before denaturation-based performance declines (Ang et al. 2021).

The results of this study indicate that the hybrid chitinase enzyme originating from B. subtilis and S. marcescens demonstrates elevated chitinase activity in comparison to the separate strains. This indicates that the hybrid strain holds greater promise for chitin degradation, presenting potential benefits across diverse industries. The ideal pH for chitinase activity was identified to be around 7, consistent with previous research findings (Garrett et al. 2012; Wu et al. 2023).

In conclusion, the study successfully identified bacterial isolates, namely B. subtilis and S. marcescens, using 16S rRNA gene sequencing. These isolates were then employed to produce and purify chitinases, showcasing their potential applications in agriculture and biotechnology. Hybrid chitinases, constructed by combining functional domains from the chitinases of both bacterial species, significantly outperformed their native counterparts in catalytic efficiency and antifungal activity. The hybrid enzyme (H-chi) exhibited superior enzymatic properties with higher activity, substrate affinity, and catalytic efficiency as demonstrated by kinetic parameters such as Km, Vmax, and kcat/Km ratios. Structural and molecular docking analyses revealed that the hybrid chitinase’s enhanced performance could be attributed to a robust hydrogen bonding network and improved binding affinity to chitin substrates. The improved antifungal activity of H-chi was evident, showing nearly double the inhibition values against fungi such as C. albicans, A. solani, and F. oxysporum compared to non-hybrid chitinases. These findings underscore the potential of the hybrid chitinase as a potent antifungal agent and its broader applicability across various industries, including waste management and biofuel manufacturing, due to its superior catalytic and antifungal properties. This study paves the way for further research and development in chitinase enzyme engineering for enhanced agricultural, biotechnological, and therapeutic applications.