CHITINASE CHRONICLES: UNVEILING EVOLUTION, MECHANISMS, CLONING, AND APPLICATIONS

be used to break down chitin in agricultural and industrial waste to produce biofuels. Chitinases can also be used to develop new anti-fungal drugs to treat fungal infections. In the food industry chitinases can be used to extract protein from shrimp and other crustacean shells, that can be used as a food ingredient. Further, chitinases can be used in the textile industry to improve the texture of the fabric and waste management strategies. Overall, chitinases have a wide range of potential applications in various industries, and ongoing research is exploring new applications for this versatile enzyme. Conceivable outcomes of a few possible utilizations of chitinase s make it an intriguing objective polymer for protein engineering. Hence, this article focuses on properties of chitinase , its mechanism of action, cloning strategy for commercial production and various associated applications. Here, in-silico agarose gel analysis of the PCR product with 1Kb maker has been carried out after cloning.


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Chitinase holds significant industrial applications due to its ability to break down chitin, a sturdy biopolymer found in the exoskeletons of insects and crustaceans.

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Chitinase also plays a pivotal role in waste management by aiding in the degradation of chitin-rich materials like shrimp shells, contributing to environmentally friendly waste disposal.

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Chitinases versatility and eco-friendly nature make it a valuable enzyme in diverse industries.

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Chitinase TA cloning is significant as it enables the efficient insertion of PCR-amplified DNA fragments into vectors.It simplifies molecular biology research, aiding in gene manipulation, protein expression, and functional studies.Chitin, the second most ubiquitous polysaccharide (after cellulose) is found in the shells of crustaceans, insects, and some fungi and in the internal structures of many vertebrates.Chitinases catalyze the cleavage of β-1,4 glycosidic linkages in chitin and contribute to the generation of carbon and nitrogen in the ecosystem.Chitinases can be used to control pests and diseases in plants.Chitinases can also enhance plant growth by breaking down chitin in the soil and making nutrients more available to plants.Chitinases can be used to break down chitin in agricultural and industrial waste to produce biofuels.Chitinases can also be used to develop new anti-fungal drugs to treat fungal infections.In the food industry chitinases can be used to extract protein from shrimp and other crustacean shells, that can be used as a food ingredient.Further, chitinases can be used in the textile industry to improve the texture of the fabric and waste management strategies.Overall, chitinases have a wide range of potential applications in various industries, and ongoing research is exploring new applications for this versatile enzyme.Conceivable outcomes of a few possible utilizations of chitinases make it an intriguing objective polymer for protein engineering.Hence, this article focuses on properties of chitinase, its mechanism of action, cloning strategy for commercial production and various associated applications.Here, in-silico agarose gel analysis of the PCR product with 1Kb maker has been carried out after cloning.
The present article focusses on the importance and various environmental applications.The article also depicts industrial applications of chitinase including its commercial production.The phylogenetic analysis of chitinase has been conducted to retrieve the vulnerability and ancestral homology.An effective cloning strategy has been designed for commercial production of chitinase.
Several research articles on chitinase effectivity, mechanism of action etc., are available but none of them has its ancestral study and an experimental organism for study.To understand a particular biomolecule, one should consider an experimental organism, so that complete ancestral, structural, and analytical studies can be carried out.A considerable research gap was identified regarding chitinase categories, ancestral relationship, and commercial production.To study the said research gap, Bacillus thuringiensis was used as an experimental organism and the findings are illustrated in this article.

CLASSIFICATION OF CHITINASE: TYPES AND FAMILIES
Endochitinases and exochitinases are the two major subcategories of chitin-lytic enzymes.Exochitinases are additionally grouped in two classes, the enzymes chitobiosidases, which catalyze the gradual release of di-acetylchitobiose from the N-terminal non-reducing end, and N-acetylglucosaminidases, which break down the oligomeric products using endochitinases into N-acetyl glucosamine (GlcNAc) monomers

EVOLUTIONARY ANALYSIS OF BACILLUS THURINGIENSIS (INSECTICIDAL) CHITINASE
The relevance of evolution to microbial waste control agents primarily depends on two wide-ranging categories: effectiveness and risk.The impact of microevolution on chitin waste management efficacy has focused on the evolution of chitin metabolizing microflora (Cory and Franklin 2012; Chakravarty and Edwards 2022).The yearly chitin production in aquatic environment is around 1011 tons (Das et al., 2012;Senevirathna et al., 2022).Chitinases synthesized by chitin-lytic bacteria degrades chitin into significant pharmaceutical end products like, N-acetyl glucosamine and chito-oligosaccharides, which are feasible substitutes of chemical methods presently being employed to achieve this goal.Enzymatic hydrolysis is the greatest solution to this issue because chemical-based remediation can cause environmental deterioration.Bt-Chitinase usage in chitin waste management approaches, like chitin augmentation to inundate entomological control, with due consideration of chitinase evolutionary principles that may influence the success rate of above strategies.Moreover, the byproducts of chitinases action over chitin are water soluble possessing anti-cancerous, anti-microbial and immuno-boosting activities (Kim et al., 2022).To perform evolutionary study, the chitinase amino acid sequence of Bacillus thuringiensis (Bt) is retrieved from genbank of NCBI with accession number ABO63962.1, the 3D structure of which is represented in figure 2. The evolutionary history (figure 3) was inferred by using MEGA-X software through Maximum Likelihood (ML) method based on the Jones-Taylor-Thornton (JTT) matrix-based model (Jones et al., 1992).The JTT model is based on empirical amino acid exchange matrices calculated from protein alignment databases that consider the average amino acid patterns of the set of data under study.The ML method is employed for generation of dendrogram and estimation amino acid substitution for any sort of microbial species.The best phylogeny and branch lengths were discovered together with the amino acid substitute matrix that produced the maximum probability for an aligning of sequences linked by single phylogenetic tree.By utilizing all the information included across the sequences at all homology levels and employing a model which specifically permits multiple modifications to take place on a single branch at any location inside an alignment.This ML technique overcomes the challenges associated with the counting approaches.The ML estimation method utilizes sequence data information more efficiently with no systematic error and generates a single phylogenetic tree.The evolutionary history of the examined taxa is represented by the bootstrap consensus tree estimated from 500 repetitions (Felsenstein 1985).Branch collapse occurred for partitions repeated in fewer than 50% of bootstrap repetitions.In the bootstrap test, the proportion of duplicate trees in which the linked taxa clustered together is displayed next to the branches (Felsenstein 1985).The analysis involved 51 amino acid sequences.All positions with less than 95% site coverage were eliminated.There was a total of 674 positions in the final dataset.Evolutionary analyses were conducted in MEGA-X (Kumar et al., 2018).Due to differences in amino acid position and number, the bootstrap values have a short range of variations.The dendrogram also depicts that the GH family of chitinase strongly relates with the Bacillus species and subspecies.Plant synthesizes chitinases as PRP, that regulates plant self-defense in response to phytopathogens attack, or by elicitors contact, such as chito-oligosaccharides (Singh et al., 2021).In comparison to the chitinases produced by insects, those produced by plants frequently have lower molecular weights.Numerous environmental issues are linked to the use of chemicals to remove plant diseases.The two primary culprits behind plant diseases are fungal and insect infestations.Nevertheless, chitinase oversees hydrolyzing chitin because it is the main structural component of insects and fungi.Thus, there is a great deal of interest in chitinase synthesis to produce biopesticides or defense proteins, such as in genetically modified plants, as well as antibacterial compounds (Khokhani et al., 2021).Although there is no apparent connection between the production of chitinase and the generation of oxygen radicals (ROS), various studies have shown that the two processes cooperate to protect plant vegetation against anthropogenic effects.

BACTERIAL CHITINASES
During the previous years, chitinases have gained increased consideration because of its wide applications (Table 1).Chitin is broadly dispersed in nature as a major component of crab's shell and shrimps, exoskeletons of insects, and cell wall of various fungi.The structural similarity of chitin with cellulose (presence of acetamide group (NH.CO.CH3) at C-2 position in chitin), makes it highly vulnerable like cellulose in nature.

THE TA CLONING STRATEGY FOR COMMERCIAL PRODUCTION OF CHITINASE
To predict the industrial production of chitinase enzyme Snap gene tool was used.
To perform the desired task reverse translation of the amino acid sequence (ABO63962.1)was carried out.The reverse translation was performed using Uniprot online tool that resulted in ssDNA.The ssDNA was then replicated to dsDNA by using Snap gene and then the resulted dsDNA was cloned by TA cloning method using Snap gene tool.At first the template (dsDNA) specific primers were designed having Tm -60 0 C and the PCR was performed using forward primer -AGATCACAAAAATTTACACTGCTGCTGCT (29mer) and reverse primer -TTGCTTCCCAGCCGCC (16mer).The PCR performance indicates that the desired inserted sequence can be identified by PCR after the TA cloning execution invitro.Now, the desired dsDNA sequence was inserted into double stranded pT7Blue T-vector of 2888 bp size that resulted in 3763 bp of recombinant vector (figure 5).The complete cloning history is given in figure 6.

USES OF CHITINASES
The chitinase mediated chitin hydrolysis produces such monomers that have significant industrial importance.Chitinases can be a vital tool in waste management as they can break down chitinous waste into smaller, simple molecules that can enter rivers in a safe manner therefore solving environmental problems (Rathod et al., 2015).In the food industry chitinases have a significant

PHARMACEUTICAL USES OF CHITINASES
In the pharmaceutical sector, chemotherapeutic medications, and allergies, chitinase serve a variety of purposes.Proteins with chitinase activity have been found to be biomarkers for cutaneous allergies and asthma in humans.Moreover, the increased immunological activity of Th-2 cells in the bronchioles is directly related to asthma.Therefore, scaling up is challenging without complete knowledge of the necessary characteristics.Not much research has been conducted using airlift and bubble column reactors.Biological systems do not assemble chitin, indicating that the main factor governing chitin's own degeneration and turnover is its de-novo production.However, the prospects for chitin's synthesis of new biomass or its transformation into inorganic components are very different.There is little knowledge about the variables governing this discrepancy.The structure of the substrates and bacterial population that uses chitin may also affect the substrate's preference for hydrolyzed metabolites.Habitat structure may influence the overall characteristics of the inheriting chitin-using population and may also influence the pace of chitin mineralization.This might have significant ramifications for carbon (C) and nitrogen (N) cycling in food webs i.e., via carbon or nitrogen removal because of mineralization and volatilization.

FUTURE PROSPECTS
Chitinase as a hydrolyzing enzyme has various applications in environmental, therapeutic, and industrial biotechnology sectors.High yield or improved catalytic activity are two ways that chitinases might be used economically and practically.
Chitinase engineering research is mostly focused on increasing its catalytic activity.Site-directed mutagenesis is a significant strategy that allows the mutation of specific amino acid moieties in chitin-binding catalytic site.For instance, the glutamic acid found in the active region of chitinase contributes significantly to the catalytic efficiency by serving as a proton contributor for breaking glycosidic bonds.Therefore, any modification close to glutamate improves the effectiveness of the enzyme.Another approach can be directed evolution.Seeds that have been chitinase-treated are found to be more resistant to fungal infestations.To generate chitinases with wide activity, directed evolution might be investigated.The function of fungal chitinases in their interaction with plants, however, is not well understood.To comprehend the mechanism and function of fungal chitinase during plant pathogenesis, further study is thus necessary.
Chitinase produced by microbes and insects, has numerous applications in waste management, SCP production, biocontrol, etc.The use of chitinase as a biocontrol agent in agriculture has important implications since it offers a safe substitute for hazardous chemical-based pest and insects control agents.
For industrial scale chitinase production, submerged fermentation is the preferred method, producing higher yields; however, this method needs more investigation, particularly on heat transfer and downstream processing issues, prior to it being considered commercially viable.
Immobilized chitinase offers great potential by reducing the time and cost of waste elimination.But identifying a perfect cross-linker to develop immobilized chitinase enzyme formulation remains a major difficulty.Due to the wide range of applications the demand for chitinases is anticipated to increase soon.

Figure 1
Figure 1 Various industrial applications of Chitinase (Taokaew and Kriangkrai 2023; Mahajan et al., 2023) Chitinases hydrolyze -1,4 linkage of N-acetyl glucosamine present in chitin chains of different sizes (20 kDa to 90 kDa) (Bhattacharya et al., 2007; Daulagala 2021).Glycoside hydrolases, a prevalent enzyme hydrolyzes glycosidic bond present between two carbohydrates molecules or a carbohydrate having non-carbohydrate part.Due to clear correlation between amino acid sequences and folding, glycoside hydrolases have been categorized into several families.The Glycoside hydrolases classification explains structural characteristics of enzymes and aids in the discovery of evolutionary connections.Additionally, this classification offers a quick method for determining the relationships and structural details between members of enzyme family and their associated substrate specificity (Cantarel et al., 2009; Schomburg and Schomburg 2010).

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Nayak et al., 2021).Chitinases from different species are classified into families 18, 19, and 20 based on similarities in amino acid composition (Henrissat 1999; Franceus et al., 2021).Chitinases from viruses, bacteria, fungi, mammals, and some plant species belongs to family 18.Family 18 is made up of many conserved amino acids repetition units with 8 parallel sheets that create a barrel-shaped down-helix, which in turn forms a ring that faces outward (Gooday 1999; Singh et al., 2021).Most plant chitinases beside Streptomyces chitinases are members of family 19 (Hart et al., 1995).Family 18 and family 19 Chitinases have different 3D structures due to varied amino acid sequences.They have therefore developed from several forebears, according to this.The enzyme Nacetylglucosaminidases from family 20, is present in humans, fungi, and bacteria.Chitolectins is another protein family (GH18) that includes chitinases and their homologous proteins.However, they still include extremely conserved residues necessary for attachment of oligosaccharide and 3D structural stability.Chitinolectins lack the essential active-site residue (glutamate), which supplies the proton needed for hydrolytic activity.Chitinases are often categorized into either GH18 or GH19 glycoside hydrolase families, each of which has a unique structure and catalytic process (Ohno et al., 1996; Chen et al., 2020).The plants, nematodes, and certain bacteria were found to have the majority of GH19 chitinases (Grover 2012).Recent studies indicate that the GH48 and GH20 protein families also contain chitinase activity (Kubota et al., 2004; Fujita et al., 2006; Orlando et al., 2021).By hydrolyzing N-acetylglucosamine, the N-acetyl-Dglucosaminidases of GH20 family breakdown chitin (GlcNAc) (Mark et al., 2001).Chitinase family 18 has a lot of repeating conserved amino acids.It is made up of an eight-stranded core of parallel sheets, a barrel oriented downward with helices, and ultimately a ring facing outward (Gooday 1999).The transglycosylation products of Trichoderma harzianum Chit42, Chit33 and Aspergillus fumigatus ChiB1 has already been reported (Martinez et al., 2012).The amino acid sequence of enzyme chitinases, belonging to two separate families are not identical, including their entirely different 3D structures and molecular processes.They most likely descended from several forebears in this way.The GH family 18 chitinases possibly catalyze trans-glycosylation processes (Orlando et al., 2021).

Figure 2
Figure 2 3D structure of Bacillus thuringiensis chitinase enzyme retrieved from genbank of NCBI

Figure 3
Figure 3 Phylogenetic tree (Neighbour-joining) based on Chitinase amino acid sequences of Bacillus thuringiensis (Bt), showing closest relationship with and is further detachable by signal peptidases once the protein has passed across the membrane.The chitinase that lack a secretory signal sequence are demonstrated to be intracellular, and their role in morphogenesis is expected(Jeong et al., 2023).Apart from N-terminal signal peptide region and catalytic domain, the structure of some fungal chitinases reported to have ser/thr rich region and chitin-binding domain, such as endochitinase of S. cerevisiae, CTS1, K. lactis KlCts1p, T. reesei Chi18-13 and Chi18-16 and R. ologosporus chitinase I and chitinase II (Koutb et al., 2021).To produce mature protein, the ser/thr rich region of fungal chitinase is often glycosylated post-translationally.MECHANISM OF CHITINASE ACTION ON CHITINChitinases chitodextrinase, glucosaminidase, glycanohydrolase are hydrolytic enzymes that break down glycosidic bonds of chitin(Singh et al., 2021;Abulikemu et al., 2021).As discussed above, endochitinase and exochitinases are the two primary groups of chitin-lytic enzymes.The group of chitinases known as endochitinases randomly cleaves at internal chitin polymer sites to produce low molecular weight multi-subunit glucosamine residues (Rathod et al., 2015).The mechanism of chitinase action to derive fructose-6-phosphate that can be utilized as energy source is given in figure4.

Figure 5 Figure 6
Figure 5 In silico TA cloning of the reverse translated dsDNA of chitinase amino acid sequence (ABO63962.1).The dsDNA is inserted into the pT7Blue T-vector expression vector where the red region indicates the gene coding for chitinase, and the black colored region represents the vector.The inserted dsDNA sequence under control of lac operator with T7 promotor.The various restriction sites are also indicated with their restriction enzyme name

Figure 7
Figure 7 Image of Agarose Gel Electrophoresis (AGE), with 1Kb marker, 1% agarose gel in 1x TBE buffer AGRICULTURAL USES OF CHITINASES Chitinases act as biocontrol agent in plants as a part of its defense mechanism.The Phyto-Chitinase acts against fungal invaders and harmful insects having chitinous exoskeletons (Banerjee and Mandal 2019).Due to lack of a sophisticated immune system like humans, plants depend on inherited defense mechanisms like chitinases for protection against phyto-pathogens (Dukare et al., 2021).A lot of research on overexpressing chitinases was conducted previously in the transgenic plants to protect them from fungal pathogens, as Chitinase can break down the pathogens cell wall which is made-up of its substrate chitin (Prasad et al., 2013; Moosa et al., 2018; Kumar et al., 2018; Eslahi et al., 2021; Yang et al., 2020).This has major significance in agriculture because crop losses due to pathogen attack can cost 200-300 billion US dollars annually (Thakur et al., 2021).Most methods to protect crops involve chemicals like pesticides or fungicides which are harmful to living organisms and the environment, so chitinase offers a safe and natural mode of protection.It is also important to consider that crops like tobacco, tomatoes, cabbage, etc., can be saved that are usually affected by fungal pathogens.A recent study suggests that beside chemical pesticides, chitinase has demonstrated its ability to effectively suppress a range of harmful fungi and insect pests (Mahajan et al., 2023).

Table 1
Bacterial sources of chitinase and their properties Bacteria utilize chitinase to break down GlcNAc-containing macromolecules (chitin) to access their source of nutrients.The development of many marine bacteria ecologically depends on the chitinase activity.Chitinases produced by bacteria range in size from 20 to 60 kDa, which is smaller than insect chitinases (40 to 85 kDa) and comparable to plant chitinases (25 to 40 kDa) (

Kotb et al., 2023
(Kotb et al., 2023)o depend on the bacteria from which they were derived.Microbial chitinases normally work in a wide variety of pH and temperature settings(Kotb et al., 2023).The optimal temperatures for the endochitinases isolated from S. violaceusniger and S. thermoviolaceus OPC-520 are 28 0 C and 80 0 C, respectively.While the chitinase isolated from Stenotrophomonas maltophilia C-3 has a pH range of 4.5 to 5.0 and the chitinase of S. thermoviolaceus OPC-520 has a high pH range of 8.0 to 10.0 (

Nayak et al., 2021). FUNGAL CHITINASE Fungi
(Deng et al., 2007)016)rades chitin for its own defense for survival and to obtain nutrients saprophytically(Homthong et al., 2016).Fungal chitinase is synthesized from chitinase genes, that are induced by the presence of chitin(Deng et al., 2007).Moreover, excessive varieties of chitinase are found among fungal species, producing different chitinase having distinct catalytic properties (Table2).

Table 2
Fungal Chitinase and Their Activities Nakamura et al., 2021)in the amino acid arrangement of the catalytic site chitinases were categorized under the glycosyl hydrolase system (Henrissat 1999;Nakamura et al., 2021).Because they are found in bacteria, fungi, yeast, viruses, plants, and mammals, the family 18 chitinases have a diverse evolutionary history.Members of family 19 are nearly entirely found in plants (Banerjee and Mandal 2019).In the glycohydrolase family of proteins, family 18, has the majority of fungal chitinases (Jiménez-Ortega et al., 2022).Chitinases from family 18 have a multi-domain organization as one of their traits (Jiménez-Ortega et al., 2022).The N-terminal signal peptide region, catalytic domain, ser/thr-rich region, chitinbinding domain, and C-terminal expansion region are the five domains (regions) that make up a typical fungal chitinase structure.The final three domains are absent from the majority of fungal chitinases.Because naturally occurring chitinases lacking these domains (regions) are nevertheless enzymatically active, it suggests that these domains are not necessary for chitinase activity.May these domains help stabilize the enzyme's overall structure and catalytic site.Each domain of the fungal chitinase structure serves a different metabolic purpose.Except for R. oligosporus chitinase III, (Takaya et al., 1998) and T. harzianum Chi18-2, Chi18-3, and Chi18-7 (Seidl-Seiboth et al., 2014), all the decoded fungal chitinases have a signal peptide added before the N-terminal region of the final protein (Sharma et al., 2022).The signal peptide facilitates the enzyme's secretion

Essghaier et al., 2010; Essghaier et al., 2021).
(Ibrahim et al., 2016)blicized that Planococcus rifitoensis, chitinase was used as a biocontrol agent for fungus called Botrytis cinerea (grey mould) which decay citrus fruits (Also, research has demonstrated that several microbial populations employ chitinases as an energy source (Gomaa et al., 2021).If fish waste from the sea food business enters rivers directly, it becomes a dangerous pollutant because it contains a lot of chitin.It raises BOD and requires a lot of demineralization, including acidic activity, as well as deproteinization (Suryawanshi and Eswari 2021).Recombinant chitinases reduce river pollution by creating chito-oligomers from chitinous waste, that are employed in a variety of biotechnological applications.Chito-oligomers are non-toxic, water-soluble substances that have antibacterial and anticancer properties as well as the ability to boost animal immunity(Ibrahim et al., 2016).Additionally, chitinase turns chitinous waste into biofertilizers, boosting crop productivity (

Mishra et al., 2016; Leite et al., 2021).
(Shao et al., 2020)2)ed in an indirect manner to estimate the biomass levels of fungi in soil.High amounts of chitinase in the soil indicate high quantities of fungi there(Sharma et al., 2022).When examining the fungal populations in the necessary soil environment, this factor may be crucial (Dukariya and Kumar 2020).In the food industry, tannase is used to break down the tannins in fruit juices like pomegranate, cranberry, etc.The fungus Aspergillus niger produces tannase, which binds to its cell wall and reduces the enzyme's total production(Shao et al., 2020).The cell wall of this fungus may also be broken down using chitinase, which could increase the amount of tannase produced (

Barthomeuf et al., 1994; Gezaf et al., 2021).
In a recent investigation of chitinolytic activity of chitinase Stenotrophomonas maltophilia portrayed the highest enzymatic activity than other selected bacterial isolates (

Shekhar et al., 2006; Sharma et al., 2022).
An earlier (2001)study exposed a link that a chitinase-likeprotein upregulates CD4+ Th-2 cells in mouse due to a specific interleukin signaling (It was the first time that an unknown link of chitinase with inflammation in the bronchioles due to asthma was explored and established (

Nayak et al., 2021; Sharma et al., 2022).
, physiological roles and it can be re-designed to utilize chitin for generating novel products of industrial use.Looking forward, chitinase has many significant applications in agricultural sector, industries, and pharmaceuticals.So, it is worth producing different chitinases at a reasonable cost, using recombinant DNA technology.Their use as biological control agents against phyto-pathogens is by far their considerable successful application, but further investigation will bring out their complete potential.Recombinant techniques can manufacture chitinases in large quantities for commercial use.Protein engineering might be used to create chitinases with the necessary functionality to satisfy the demands of chitinases for treating various disorders.It is critical to choose reactors carefully that promote the enhanced synthesis of chitinases with the right parameters.It is mandatory to use appropriate carbon sources since they have a significant effect on the production of enzymes.Aeration and agitation rate have little bearing on the synthesis of chitinase.
(Shekhar et al., 2006;Regev et al., 1996)itinases also offer supportive statistics for their potential use as a biomarker in Th-2 associated alveolar infection, like that of TKL-40 and AMCase(Shekhar et al., 2006;Regev et al., 1996).CONCLUSIONThe diverse family of chitinases have a unique molecular geometry, substrate selectivity, and catalytic mechanism.It is important to study and identify the chitinase-substrate specificity, as it could reveal its relationship with specific substrate types