Chitinases are essential enzymes that play a significant role in various biological processes by catalyzing the degradation of chitin, a key component of fungal cell walls. Due to their potential applications in agriculture, pharmaceuticals, and industry, chitinases have garnered much attention in recent years (Monge et al. 2018; Pentekhina et al. 2020; Tran et al. 2022).
Bacillus subtilis and Serratia marcescens are two bacteria known to produce chitinases with inherent antifungal properties. These enzymes have demonstrated efficacy in inhibiting the growth of pathogenic fungi, making them potential candidates for exploring novel antifungal treatments (De Medeiros et al. 2018; Cd et al. 2021; Dikbaş et al. 2023).
The use of chitinase enzymes, which break down chitin in fungal cell walls, is a promising approach. These enzymes are produced by bacteria such as Bacillus subtilis and Serratia marcescens and have been shown to have antifungal properties. By combining functional domains from different bacterial chitinases, the effectiveness of fungal inhibition can be increased through the creation of hybrid enzymes (Oranusi and Trinci 1985; Senol et al. 2014).
Chitinases are essential enzymes that play a significant role in various biological processes by catalyzing the degradation of chitin, a key component of fungal cell walls. Due to their potential applications in agriculture, pharmaceuticals, and industry, chitinases have garnered much attention in recent years (Monge et al. 2018; Pentekhina et al. 2020; Tran et al. 2022).
B. subtilis and S. marcescens are two bacteria known to produce chitinases with inherent antifungal properties. These enzymes have demonstrated efficacy in inhibiting the growth of pathogenic fungi, making them potential candidates for exploring novel antifungal treatments (Carter-House et al. 2020; Okay and Alshehri 2020).
The objective of this study was to create genetically modified hybrid chitinase enzymes with improved antifungal properties by combining functional domains from B. subtilis and S. marcescens chitinases. To achieve this, we cloned the chitinase genes from both bacteria and used overlap extension PCR to fuse them together. The resulting hybrid constructs were expressed in Escherichia coli, enabling the production of recombinant hybrid chitinases.
Identifying bacterial isolates is crucial as it offers insights into their origin, potential pathogenicity, and unique characteristics. This knowledge enhances our understanding of how microorganisms interact with their environment. Moreover, identifying the strains is essential for designing and interpreting future experiments involving the isolates. The precise identification of bacterial isolates was essential in this study to comprehend their biological characteristics, potential for causing disease, and ecological importance. To accomplish this, the bacterial isolates underwent 16S rRNA gene sequencing, a method renowned for its strong conservation and inclusion of both variable and conserved regions (Iskandar et al. 2021; Ahmed et al. 2022). The sequencing technique was successful in accurately identifying the bacterial isolates at the species level. These identified isolates were then utilized to produce and purify chitinases, which have potential uses in agriculture and biotechnology. The precise identification of the isolates enabled the successful identification and cloning of the appropriate chitinase genes, facilitating the expression and evaluation of the chitinases' activity (Nguyen Hoang et al. 2022).
The BLAST analysis of the B. subtilis isolate demonstrated 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 finding further supports the identification of the isolate as B. subtilis. Likewise, the phylogenetic analysis of the S. marcescens isolate indicated 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 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 gene 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 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, 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-ChiA, 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-Chi, 6.56 for Sm-ChiA, 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 x 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.
The findings indicate that the hybrid chitinase (H-Chi) 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.
To assess the antifungal properties of the purified recombinant chitinases, an agar diffusion assay was conducted against the plant pathogen F. oxysporum. The chitinases were placed in wells on plates containing F. oxysporum conidia, and the inhibition zones were measured after incubation. The findings revealed that the hybrid chitinase displayed the largest inhibition zone, measuring 22 mm, surpassing the Sm-Chi (16 mm) and Bs-Chi (12 mm) chitinases. The results of this study demonstrate that the hybrid chitinase exhibits strong antifungal activity against F. oxysporum. The larger zone of inhibition indicates that the hybrid chitinase is more efficient in suppressing the growth of F. oxysporum compared to the other chitinases evaluated. This finding is significant because F. oxysporum is a widely encountered plant pathogen that causes numerous plant diseases.
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 the 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 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 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, this study investigated the antifungal potential of genetically modified hybrid chitinase enzymes derived from B. subtilis and S. marcescens. Hybrid chitinase enzymes with enhanced antifungal activity were created by combining functional domains from the native chitinases produced by B. subtilis and S. marcescens. The chitinase genes were cloned, fused together using PCR, and expressed in E. coli. The recombinant enzymes were purified, and their molecular weights and isoelectric points were confirmed. Antifungal assays showed that the hybrid chitinases inhibited the growth of the fungus F. oxysporum more effectively than the native enzymes. Also, this study demonstrated the antifungal activity of purified recombinant chitinases against F. oxysporum using an agar diffusion assay. The hybrid chitinase exhibited the largest inhibition zone, surpassing the other chitinases tested. Furthermore, 3D homology modeling of the chitinases provided valuable structural insights into their antifungal activity. These findings contribute to our understanding of chitinases as potential candidates for the development of antifungal agents in plant protection strategies. Future studies could focus on elucidating the precise mechanisms through which chitinases inhibit fungal growth and explore their application in agricultural practices.