Insoluble Chitin Research Articles

Enhanced degradation of insoluble chitin: Engineering high-efficiency chitinase fusion enzymes for sustainable applications

N-acetyl-D-glucosamine and its dimer are degradation products of chitin waste with great potential in therapeutic and agricultural applications. However, the hydrolysis of insoluble chitin by chitinases remains a major bottleneck. This study investigated the biochemical properties and catalytic mechanisms of PoChi chitinase obtained from Penicillium oxalicum with a focus on enhancing its efficiency during the degradation of insoluble chitin. Recombinant plasmids were engineered to incorporate chitin-binding (ChBD) and/or fibronectin III (FnIII) domains. Notably, PoChi-FnIII-ChBD exhibited the highest substrate affinity (Km = 2.7 mg/mL) and a specific activity of 15.4 U/mg, which surpasses those of previously reported chitinases. These findings highlight the potential of engineered chitinases in advancing industrial biotechnology applications and offer a promising approach to more sustainable chitin waste management.

Engineering a recombinant chitinase from the marine bacterium Bacillus aryabhattai with targeted activity on insoluble crystalline chitin for chitin oligomer production

Chitin, an abundant polysaccharide in India, is primary by-product of the seafood industry. Efficiently converting chitin into valuable products is crucial. Chitinase, transforms chitin into chitin oligomers, holds significant industrial potential. However, the crystalline and insoluble nature of chitin makes the conversion process challenging. In this study, a recombinant chitinase from marine bacteria Bacillus aryabhattai was developed. This enzyme exhibits activity against insoluble chitin substrates, chitin powder and flakes. The chitinase gene was cloned into the pET 23a plasmid and transformed into E. coli Rosetta pLysS. IPTG induction was employed to express chitinase, and purification using Ni-NTA affinity chromatography. Optimal chitinase activity against colloidal chitin was observed in Tris buffer at pH 8, temperature 55°C, with the presence of 400 mM sodium chloride. Enzyme kinetics studies revealed a Vmax of 2000 μmole min−1 and a Km of 4.6 mg mL−1. The highest chitinase activity against insoluble chitin powder and flakes reached 875 U mg−1 and 625 U mg−1, respectively. The chitinase demonstrated inhibition of Candida albicans, Fusarium solani, and Penicillium chrysogenum growth. Thin Layer Chromatography (TLC) and LC-MS analysis confirmed the production of chitin oligomers, chitin trimer, tetramer, pentamer, and hexamer, from chitin powder and flakes using recombinant chitinase.

The “life-span” of lytic polysaccharide monooxygenases (LPMOs) correlates to the number of turnovers in the reductant peroxidase reaction

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that degrade the insoluble crystalline polysaccharides cellulose and chitin. Besides the H2O2 cosubstrate, the cleavage of glycosidic bonds by LPMOs depends on the presence of a reductant needed to bring the enzyme into its reduced, catalytically active Cu(I) state. Reduced LPMOs that are not bound to substrate catalyze reductant peroxidase reactions, which may lead to oxidative damage and irreversible inactivation of the enzyme. However, the kinetics of this reaction remain largely unknown, as do possible variations between LPMOs belonging to different families. Here, we describe the kinetic characterization of two fungal family AA9 LPMOs, TrAA9A of Trichoderma reesei and NcAA9C of Neurospora crassa, and two bacterial AA10 LPMOs, ScAA10C of Streptomyces coelicolor and SmAA10A of Serratia marcescens. We found peroxidation of ascorbic acid and methyl-hydroquinone resulted in the same probability of LPMO inactivation (pi), suggesting that inactivation is independent of the nature of the reductant. We showed the fungal enzymes were clearly more resistant toward inactivation, having pi values of less than 0.01, whereas the pi for SmAA10A was an order of magnitude higher. However, the fungal enzymes also showed higher catalytic efficiencies (kcat/KM(H2O2)) for the reductant peroxidase reaction. This inverse linear correlation between the kcat/KM(H2O2) and pi suggests that, although having different life spans in terms of the number of turnovers in the reductant peroxidase reaction, LPMOs that are not bound to substrates have similar half-lives. These findings have not only potential biological but also industrial implications.

The molecular mechanism for carbon catabolite repression of the chitin response in Vibrio cholerae.

Vibrio cholerae is a facultative pathogen that primarily occupies marine environments. In this niche, V. cholerae commonly interacts with the chitinous shells of crustacean zooplankton. As a chitinolytic microbe, V. cholerae degrades insoluble chitin into soluble oligosaccharides. Chitin oligosaccharides serve as both a nutrient source and an environmental cue that induces a strong transcriptional response in V. cholerae. Namely, these oligosaccharides induce the chitin sensor, ChiS, to activate the genes required for chitin utilization and horizontal gene transfer by natural transformation. Thus, interactions with chitin impact the survival of V. cholerae in marine environments. Chitin is a complex carbon source for V. cholerae to degrade and consume, and the presence of more energetically favorable carbon sources can inhibit chitin utilization. This phenomenon, known as carbon catabolite repression (CCR), is mediated by the glucose-specific Enzyme IIA (EIIAGlc) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). In the presence of glucose, EIIAGlc becomes dephosphorylated, which inhibits ChiS transcriptional activity by an unknown mechanism. Here, we show that dephosphorylated EIIAGlc interacts with ChiS. We also isolate ChiS suppressor mutants that evade EIIAGlc-dependent repression and demonstrate that these alleles no longer interact with EIIAGlc. These findings suggest that EIIAGlc must interact with ChiS to exert its repressive effect. Importantly, the ChiS suppressor mutations we isolated also relieve repression of chitin utilization and natural transformation by EIIAGlc, suggesting that CCR of these behaviors is primarily regulated through ChiS. Together, our results reveal how nutrient conditions impact the fitness of an important human pathogen in its environmental reservoir.

From Chitosan to Chitin: Bio-Inspired Thin Films for Passive Daytime Radiative Cooling.

Passive radiative daytime cooling is an emerging technology contributing to carbon-neutral heat management. Optically engineered materials with distinct absorption and emission properties in the solar and mid-infrared range are at the heart of this technology. Owing to their low emissive power of about 100W m-2 during daytime, substantial areas need to be covered with passive cooling materials or coatings to achieve a sizeable effect on global warming. Consequently, biocompatible materials are urgently needed to develop suitable coatings with no adverse environmental impact. It is shown how chitosan films with different thicknesses can be produced from slightly acidic aqueous solutions. The conversion to their insoluble form chitin in the solid state is demonstrated and the conversion is monitored with infrared (IR) and NMR spectroscopy. In combination with a reflective backing material, the films show below-ambient temperature cooling capabilities with a suitable emissivity in the mid-IR region and low solar absorption of 3.1-6.9%, depending on the film thickness. This work highlights the potential of chitosan and chitin as widely available biocompatible polymers for passive radiative cooling applications.

Regulators of natural competence in Vibrio parahaemolyticus.

Vibrio parahaemolyticus can degrade insoluble chitin with the help of chitinase enzymes that generate soluble N-acetyl glucosamine oligosaccharides (GlcNAcn) to induce a state of natural competence for the uptake of extracellular DNA. In this study, we had evaluated the role of various regulatory factors such as TfoX, CytR, OpaR, and RpoS during natural transformation of V. parahaemolyticus. The results suggest that TfoX regulates natural competence via CytR in a chitin-dependent manner. CytR controls the release of GlcNAc6 from insoluble chitin and conversion of GlcNAc6 into smaller GlcNAc residues inside the periplasm by modulating the expression of endochitinase and periplasmic chitinases. In addition, CytR was also responsible for GlcNAc6-mediated upregulation of competence-related genes such as pilA, pilB, comEA, and qstR. Next, we found that the quorum sensing regulator OpaR affects the natural transformation through its regulation of extracellular nuclease Dns. The ΔopaR mutant showed increased expression of Dns, which might degrade the eDNA. As a consequence, the transformation efficiency was decreased and eDNA-dependent growth was hugely enhanced. However, when Dns-containing DASW was substituted with fresh DASW, the transformation was detectable in ΔopaR mutant and eDNA-dependent growth was less. These results suggest that the occurrence of natural transformation and eDNA-dependent growth were inversely related to each other. Lastly, the general stress regulator RpoS was required for neither quorum-sensing dependent nor chitin-dependent regulation of natural competence in V. parahaemolyticus.

Chitin degradation and its effect on natural transformation: a systematic genetic study in Vibrio parahaemolyticus.

The degradation of polymeric chitin by chitinase liberates soluble N-acetyl glucosamine oligosaccharides (GlcNAcn≥2), a source of nutrition that can also induce a state of natural genetic competence in Vibrio parahaemolyticus. This analysis revealed that among seven predicted chitinases, the synergistic action of VPA0055 (ChiA2), VP0619 (ChiB), and VPA0832 (Cdx) were essential for the robust growth and high transformation frequency on chitin. The endochitinase, ChiA2, and periplasmic chitinase, Cdx were indispensable for chitin degradation. ChiB was not essential for growth on chitin but did have an effect on the rate of chitin degradation. Interestingly, the loss of Cdx drastically reduced growth on insoluble chitin, but growth on soluble GlcNAc5/6 remained same. The utilization of GlcNAc5/6 was only inhibited when there was mutation of Cdx with the other periplasmic chitinases VP0755 and VP2486. This suggests that Cdx might also be involved in extracellular degradation of chitin, in addition to its role as a periplasmic chitinase. Moreover, the periplasmic chitin oligosaccharide-binding protein (CBP) was found to be essential for the efficient utilization of chitin. The CBP was specifically needed for the processing of GlcNAc4-6 during growth on chitin. Overall, this study provides detailed analysis of the machinery behind chitin degradation in V. parahaemolyticus.

Expression, purification, and basic properties of a novel domain structure possessing chitinase from Escherichia coli carrying the family 18 chitinase gene of Bacillus velezensis strain RB.IBE29.

Bacillus velezensis possesses numerous chitinolytic enzymes; however, not much is known about the role of its chitinase molecules. In this study, the chiA gene, which encodes a novel domain structure possessing family 18 chitinase from B. velezensis RB.IBE29, was expressed successfully in Escherichia coli BL21-CodonPlus (DE3)-RIPL using the pColdII expression vector. The recombinant protein, rBvChiA, was purified using the HisTrap FF column. Purified rBvChiA showed hydrolytic activity against insoluble chitin and bound to chitinous substrates. In addition, the purified recombinant enzyme displayed remarkable inhibition effects on the spore germination of Fusarium falciforme and the egg hatch of root-knot nematodes (Meloidogyne spp.), which are the main causes of black pepper diseases in the Central Highlands region, Vietnam. The current work results might enable further studies to develop novel chitinase A and strain RB.IBE29 as a natural fungicide and nematicide for sustainable black pepper production and other crops in the Central Highlands, Vietnam. This is the second report describing chitinase from B. velezensis based on the experimental data.

A marine chitinase from Bacillus aryabhattai with antifungal activity and broad specificity toward crystalline chitin degradation

Chitinases convert chitin into chitin oligomers and are also known antifungal agents. Chitin oligomers have numerous industrial applications. However, chitin’s crystalline nature requires pretreatment before breakdown into oligomers. In the study, a novel marine bacterium Bacillus aryabhattai is isolated from the Arabian Sea. Bacterial growth in different crystalline chitin substrates like chitin powder, chitin flakes, and colloidal chitin confirmed the chitinase presence in bacterium could act upon insoluble crystalline chitin with the fractional release of oligomers. The domain architecture analysis of the chitinase confirmed the presence of two N-terminal LysM domains which help enzyme action on crystalline chitin. Statistical optimization of media and Process parameters revealed glycerol, yeast extract, magnesium chloride, and manganese sulfate as significant media components along with colloidal chitin. The optimum process parameters such as pH 7, temperature 40 °C, inoculum size 12.5% (v/v), and inoculum age 20 hours enhanced the specific enzyme activity to ±146.2 U/mL, ±114.9 U/mL and ±175.4 U/mL against chitin powder, chitin flakes and colloidal chitin respectively, which is five to six times higher than basal level activity. The antifungal activity of chitinase against plant pathogenic fungi like Candida albicans and Fusarium oxysporum revealed a zone of inhibition with 14 mm diameter.

Molecular analysis of genes involved in chitin degradation from the chitinolytic bacterium Bacillus velezensis.

Bacillus velezensis RB.IBE29 is a potent biocontrol agent with high chitinase activity isolated from the rhizosphere of black pepper cultivated in the Central Highlands, Vietnam. Genome sequences revealed that this species possesses some GH18 chitinases and AA10 protein(s); however, these enzymes have not been experimentally characterized. In this work, three genes were identified from the genomic DNA of this bacterium and cloned in Escherichia coli. Sequence analysis exhibited that the ORF of chiA consists of 1,203bp and encodes deduced 45.46kDa-chitinase A of 400 aa. The domain structure of chitinase A is composed of a CBM 50 domain at the N-terminus and a catalytic domain at the C-terminus. The ORF of chiB includes 1,263bp and encodes deduced 47.59kDa-chitinase B of 420 aa. Chitinase B consists of two CBM50 domains at the N-terminus and a catalytic domain at the C-terminus. The ORF of lpmo10 is 621bp and encodes a deduced 22.44kDa-AA10 protein, BvLPMO10 of 206 aa. BvLPMO10 contains a signal peptide and an AA10 catalytic domain. Chitinases A and B were grouped into subfamily A of family 18 chitinases. Amino acid sequences in their catalytic domains lack aromatic residues (Trp, Phe, Tyr) probably involved in processivity and substrate binding compared with well-known bacterial GH18 chitinases. chiB was successfully expressed in E. coli. Purified rBvChiB degraded insoluble chitin and was responsible for inhibition of fungal spore-germination and egg hatching of plant-parasitic nematode. This is the first report describing the analysis of the chitinase system from B. velezensis.

Rationally engineered chitin deacetylase from Arthrobacter sp. AW19M34-1 with improved catalytic activity toward crystalline chitin

Chitin and its derivatives have anticoagulant, antimicrobial, and antioxidant properties, but the poor solubility of chitin limits its application in different fields. In this study, site-directed mutagenesis was performed to enhance the deacetylation activity of chitin deacetylases CDA from Arthrobacter (ArCE4). The mutant Mut-2-8 with Y172E/E200S/Y201W showed a 2.84- fold and 1.39-fold increase in catalytic efficiency (kcat/Km) for the deacetylation of (GluNAc)5 and α-chitin, respectively. These results demonstrated that the mutations significantly improved the activation of ArCE4 on crystalline chitin. The molecular docking study confirmed that the enhancement of catalytic efficiency is due to the extra two hydrogen bonds and one acetyl group. In summary, the activity of Mut-2-8 to insoluble chitin was significantly improved by reactional design, which is beneficial to resolve the issues of the expensive cost of the enzymes and low efficiency. Mut-2-8 exhibits potential applications in the chitosan industry.

Isolation and characterization of chitinolytic bacterium, Escherichia fergusonii AMC01 from insectivorous bat, Taphozous melanopogon.

Chitinases are capable of hydrolyzing insoluble chitin into its oligo and monomeric parts and have received increased consideration because of their wide scope of biotechnological applications. The commercial application of microbial chitinase is appealing due to the relative ease of enormous production and to meet the current world demands. This study aimed at isolation and characterization of chitin degrading bacteria from the gut of Indian tropical insectivorous black-bearded tomb bat, Taphozous melanopogon. The isolated bacterial strains were characterized through biochemical analysis and nucleic acid-based approaches by 16S ribosomal RNA amplification and sequencing. The BLAST (Basic Local Alignment Search Tool) and phylogenetic analysis showed that the bacterial strain exhibited a close resemblance with Escherichia fergusonii. The chitinolytic activity of the E. fergusonii AMC01 was identified using supplemented colloidal chitin with agar medium. Compiling all, these findings would facilitate in constructing a database and presumably promote the use of E. fergusonii AMC01 as an efficient strain for the chitinase production.

The deletion of chiMaD1, a horizontally acquired chitinase of Metarhizium anisopliae, led to higher virulence towards the cattle tick (Rhipicephalus microplus).

The first line of the Arthropods defense against infections is the hard-structured exoskeleton, a physical barrier, usually rich in insoluble chitin. For entomopathogenic fungi that actively penetrate the host body, an arsenal of hydrolytic enzymes (as chitinases and N-acetylglucosaminidases), that break down chitin, is essential. Notably, twenty-one putative chitinase genes have been identified in the genome of Metarhizium anisopliae, a generalist entomopathogenic fungus. As a multigenic family, with enzymes that, presumably, perform redundant functions, the main goal is to understand the singularity of each one of such genes and to discover their precise role in the fungal life cycle. Specially chitinases that can act as virulence determinants are of interest since these enzymes can lead to more efficient biocontrol agents. Here we explored a horizontally acquired chitinase from M. anisopliae, named chiMaD1. The deletion of this gene did not lead to phenotypic alterations or diminished supernatant's chitinolytic activity. Surprisingly, chiMaD1 deletion enhanced M. anisopliae virulence to the cattle tick (Rhipicephalus microplus) larvae and engorged females, while did not alter the virulence to the mealworm larvae (Tenebrio molitor). These results add up to recent reports of deleted genes that enhanced entomopathogenic virulence, showing the complexity of host-pathogen interactions.

Engineering a carbohydrate binding module to enhance chitinase catalytic efficiency on insoluble chitinous substrate

Development of a high-performance chitinase for efficient biotransformation of insoluble chitinous substrate would be highly valuable in industry. In this study, the chitin-binding domains (ChBDs) of chitinase SaChiA4 were successfully modified to improve the enzymatic activity. The engineered substitution variant R-SaChiA4, which had the exogenous ChBD of chitinase ChiA1 from Bacillus circulans WL-12 (ChBDChiA1) substituted for its original ChBDChiA4, increased its activity by nearly 54% (28.0 U/mg) towards chitin powder, and by 49% towards colloidal chitin, compared with the wild-type. The substrate-binding assay demonstrated that the ChBD could enhance the capacity of enzymatic hydrolysis by promoting substrate affinity, and molecular dynamics simulations indicated that this could be due to hydrophobic interactions in different substrate binding modes. This work advances the understanding of the role of the ChBD, and provides a step towards the achievement of industrial-scale hydrolysis and utilization of insoluble chitin.

Chromatographic Assays for the Enzymatic Degradation of Chitin.

Chitin is an insoluble linear polymer of β(1→4)-linked N-acetylglucosamine. Enzymatic cleavage of chitin chains can be achieved using hydrolytic enzymes, called chitinases, and/or oxidative enzymes, called lytic polysaccharide monooxygenases (LPMOs). These two groups of enzymes have different modes of action and yield different product types that require different analytical methods for detection and quantitation. While soluble chromogenic substrates are readily available for chitinases, proper insight into the activity of these enzymes can only be obtained by measuring activity toward their polymeric, insoluble substrate, chitin. For LPMOs, only assays using insoluble chitin are possible and relevant. Working with insoluble substrates complicates enzyme assays from substrate preparation to product analysis. Here, we describe typical set-ups for chitin degradation reactions and the chromatographic methods used for product analysis. Graphical abstract: Overview of chromatographic methods for assessing the enzymatic degradation of chitin.

Symbiotic chitin degradation by a novel anaerobic thermophilic bacterium Hydrogenispora sp. UUS1-1 and the bacterium Tepidanaerobacter sp. GT38

Chitin is the second most abundant organic compound in nature. Although mesophilic bacteria degrade insoluble chitin, there is a paucity of data describing degradation of insoluble chitin by anaerobic thermophilic bacteria. In this report, we screened cow manure compost for new chitin degradation systems, and identified a chitinolytic bacterial community (CBC) that showed high chitin degradation activity under thermophilic conditions, i.e., 1% (w/v) chitin powder degraded completely within 7 days at 60 °C. Metagenomic analysis revealed that the CBC was dominated by two bacterial genera from Hydrogenispora, an uncultured taxonomic group, and Tepidanaerobacter. Hydrogenispora were abundant in the early-to-mid stages of culturing with chitin, whereas the population of Tepidanaerobacter increased during the later stages of culturing. Strains UUS1-1 and GT38, which were isolated as pure cultures using the roll-tube method with colloidal chitin, N-acetyl-d-glucosamine, and glucose as carbon sources, were found to be closely related to H. ethanolica and T. acetatoxydans, respectively. Strain UUS1−1 readily degraded chitin and is the first anaerobic thermophilic chitinolytic bacterium reported, whereas strain GT38 showed no chitinolytic activity. Based on phylogenetic analysis, UUS1−1 and GT38 should be classified as novel genera and species. Zymogram analysis revealed that UUS1−1 produces at least two chitinases with molecular weights of 150 and 40 kDa. A coculture of UUS1−1 and GT38 degraded crystalline chitin faster with lower accumulation of lactate compared with UUS1−1 alone, indicating that the strains maintained a symbiotic association through assimilation of organic acids in chitin degradation and that strain GT38 consumed end-products to reduce end-product inhibition and enhance the degradation of crystalline chitin.

A fast and easy strategy for lytic polysaccharide monooxygenase-cleavable His6-Tag cloning, expression, and purification

Lytic polysaccharide monooxygenases (LPMOs) are industrially important enzymes able to enhance the enzymatic lignocellulose saccharification in synergism with classical glycoside hydrolases. Fungal LPMOs have been classified as AA9, AA11, and AA13-16 families showing a diverse specificity for substrates such as soluble and insoluble beta-glucans, chitin, starch, and xylan, besides cellulose. These enzymes are still not fully characterized, and for example this is testify by their mechanism of oxidation regularly reviewed multiple times in the last decade. Noteworthy is that despite the extremely large abundance in the entire Tree of Life, our structural and functional knowledge is based on a restricted pool of LPMO, and probably one of the main reason reside in the challenging posed by their heterologous expression. Notably, the lack of a simple cloning protocol that could be universally applied to LPMO, hinders the conversion of the ever-increasing available genomic information to actual new enzymes. Here, we provide an easy and fast protocol for cloning, expression, and purification of active LPMOs in the following architecture: natural signal peptide, LPMO enzyme, TEV protease site, and His6-Tag. For this purpose, a commercial methanol inducible expression vector was initially modified to allow the LPMO expression containing the above characteristics. Gibson assembly, a one-step isothermal DNA assembly, was adopted for the direct assembly of intron-less or intron-containing genes and the modified expression vector. Moreover, His6-tagged LPMO constructs can be submitted to TEV proteolysis for removal of the questionable C-terminal His6-Tag, obtaining a close-to-native form of LPMO. We successfully applied this method to clone, express, and purify six LPMOs from AA9 family with different regioselectivities. The proposed protocol, provided as step-by-step, could be virtually applied in many laboratories thanks to the choice of popular and commons materials.

Identification of methionine -rich insoluble proteins in the shell of the pearl oyster, Pinctada fucata

The molluscan shell is a biomineral that comprises calcium carbonate and organic matrices controlling the crystal growth of calcium carbonate. The main components of organic matrices are insoluble chitin and proteins. Various kinds of proteins have been identified by solubilizing them with reagents, such as acid or detergent. However, insoluble proteins remained due to the formation of a solid complex with chitin. Herein, we identified these proteins from the nacreous layer, prismatic layer, and hinge ligament of Pinctada fucata using mercaptoethanol and trypsin. Most identified proteins contained a methionine-rich region in common. We focused on one of these proteins, NU-5, to examine the function in shell formation. Gene expression analysis of NU-5 showed that NU-5 was highly expressed in the mantle, and a knockdown of NU-5 prevented the formation of aragonite tablets in the nacre, which suggested that NU-5 was required for nacre formation. Dynamic light scattering and circular dichroism revealed that recombinant NU-5 had aggregation activity and changed its secondary structure in the presence of calcium ions. These findings suggest that insoluble proteins containing methionine-rich regions may be important for scaffold formation, which is an initial stage of biomineral formation.

Chitinase system of Aeromonas salmonicida, and characterization of enzymes involved in chitin degradation.

The genes encoding chitin-degrading enzymes in Aeromonas salmonicida SWSY-1.411 were identified and cloned in Escherichia coli. The strain contained two glycoside hydrolase (GH) families 18 chitinases: AsChiA and AsChiB, two GH19 chitinases: AsChiC and AsChiD, and an auxiliary activities family 10 protein, lytic polysaccharide monooxygenase: AsLPMO10A. These enzymes were successfully expressed in E. coli and purified. AsChiB had the highest hydrolytic activity against insoluble chitin. AsChiD had the highest activity against water-soluble chitin. The peroxygenase activity of AsLPMO10A was lower compared to SmLPMO10A from Serratia marcescens. Synergism on powdered chitin degradation was observed when AsChiA and AsLPMO10A were combined with other chitinases of this strain. More than twice the increase of the synergistic effect was observed when powdered chitin was treated by a combination of AsLPMO10A with all chitinases. GH19 chitinases suppressed the hyphal growth of Trichoderma reesei.

Thermophilic Chitinases: Structural, Functional and Engineering Attributes for Industrial Applications.

Chitin is the second most widely found natural polymer next to cellulose. Chitinases degrade the insoluble chitin to bioactive chitooligomers and monomers for various industrial applications. Based on their function, these enzymes act as biocontrol agents against pathogenic fungi and invasive pests compared with conventional chemical fungicides and insecticides. They have other functional roles in shellfish waste management, fungal protoplast generation, and Single-Cell Protein production. Among the chitinases, thermophilic and thermostable chitinases are gaining popularity in recent years, as they can withstand high temperatures and maintain the enzyme stability for longer periods. Not all chitinases are thermostable; hence, tailor-made thermophilic chitinases are designed to enhance their thermostability by direct evolution, genetic engineering involving mutagenesis, and proteomics approach. Although research has been done extensively on cloning and expression of thermophilic chitinase genes, there are only few papers discussing on the mechanism of chitin degradation using thermophiles. The current review discusses the sources of thermophilic chitinases, improvement of protein stability by gene manipulation, metagenomics approaches, chitin degradation mechanism in thermophiles, and their prospective applications for industrial, agricultural, and pharmaceutical purposes.