High-level production of chitinase by multi-strategy combination optimization in Bacillus licheniformis.

In view of the extensive potential applications of chitinase (ChiA) in various fields such as agriculture, environmental protection, medicine, and biotechnology, the development of a high-yielding strain capable of producing chitinase with enhanced activity holds significant importance. The objective of this study was to utilize the extracellular chitinase from Bacillus thuringiensis as the target, and Bacillus licheniformis as the expression host to achieve heterologous expression of ChiA with enhanced activity. Initially, through structural analysis and molecular dynamics simulation, we identified key amino acids to improve the enzymatic performance of chitinase, and the specific activity of chitinase mutant D116N/E118N was 48% higher than that of the natural enzyme, with concomitant enhancements in thermostability and pH stability. Subsequently, the expression elements of ChiA(D116N/E118N) were screened and modified in Bacillus licheniformis, resulting in extracellular ChiA activity reached 89.31 U/mL. Further efforts involved the successful knockout of extracellular protease genes aprE, bprA and epr, along with the gene clusters involved in the synthesis of by-products such as bacitracin and lichenin from Bacillus licheniformis. This led to the development of a recombinant strain, DW2△abelA, which exhibited a remarkable improvement in chitinase activity, reaching 145.56 U/mL. To further improve chitinase activity, a chitinase expression frame was integrated into the genome of DW2△abelA, resulting in a significant increas to 180.26 U/mL. Optimization of fermentation conditions and medium components further boosted shake flask enzyme activity shake flask enzyme activity, achieving 200.28 U/mL, while scale-up fermentation experiments yielded an impressive enzyme activity of 338.79 U/mL. Through host genetic modification, expression optimization and fermentation optimization, a high-yielding ChiA strain was successfully constructed, which will provide a solid foundation for the extracellular production of ChiA.

Oschib1 gene encoding a GH18 chitinase confers resistance against sheath blight disease of rice caused by Rhizoctonia solani AG1-IA.

Sheath blight disease of rice caused by Rhizoctonia solani AG1-IA, is a major fungal disease responsible for huge loss to grain yield and quality. The major limitation of achieving persistent and reliable resistance against R. solani is the governance of disease resistance trait by many genes. Therefore, functional characterization of new genes involved in sheath blight resistance is necessary to understand the mechanism of resistance as well as evolving effective strategies to manage the disease through host-plant resistance. In this study, we performed RNA sequencing of six diverse rice genotypes (TN1, BPT5204, Vandana, N22, Tetep, and Pankaj) from sheath and leaf tissue of control and fungal infected samples. The approach for identification of candidate resistant genes led to identification of 352 differentially expressed genes commonly present in all the six genotypes. 23 genes were analyzed for RT-qPCR expression which helped identification of Oschib1 showing differences in expression level in a time-course manner between susceptible and resistant genotypes. The Oschib1 encoding classIII chitinase was cloned from resistant variety Tetep and over-expressed in susceptible variety Taipei 309. The over-expression lines showed resistance against R. solani, as analyzed by detached leaf and whole plant assays. Interestingly, the resistance response was correlated with the level of transgene expression suggesting that the enzyme functions in a dose dependent manner. We report here the classIIIb chitinase from chromosome10 of rice showing anti-R. solani activity to combat the dreaded sheath blight disease.

From Natural Microbe Screening to Sustained Chitinase Activity in Exogenous Hosts.

Genetic parts and hosts can be sourced from nature to realize new functions for synthetic biology or to improve performance in a particular application environment. Here, we proceed from the discovery and characterization of new parts to stable expression in new hosts with a particular focus on achieving sustained chitinase activity. Chitinase is a key enzyme for various industrial applications that require the breakdown of chitin, the second most abundant biopolymer on the earth. Diverse microbes exhibit chitinase activity, but for applications, the environmental conditions for optimal enzyme activity and microbe fitness must align with the application context. Achieving sustained chitinase activity under broad conditions in heterologous hosts has also proven difficult due to toxic side effects. Toward addressing these challenges, we first screen ocean water samples to identify microbes with chitinase activity. Next, we perform whole genome sequencing and analysis and select a chitinase gene for heterologous expression. Then, we optimize transformation methods for target hosts and introduce chitinase. Finally, to achieve robust function, we optimize ribosome binding sites and discover a beneficial promoter that upregulates chitinase expression in the presence of colloidal chitin in a sense-and-respond fashion. We demonstrate chitinase activity for >21 days in standard (Escherichia coli) and nonstandard (Roseobacter denitrificans) hosts. Besides enhancing chitinase applications, our pipeline is extendable to other functions, identifies natural microbes that can be used directly in non-GMO contexts, generates new parts for synthetic biology, and achieves weeks of stable activity in heterologous hosts.

Expression profiles and characterization of microRNAs responding to chitin in Arthrobotrys oligospora.

Extracellular proteases, such as chitinases secreted by Arthrobotrys oligospora (A. oligospora), play a crucial role in the process of nematode infection. However, post-transcriptional regulation of gene expression involving microRNAs (miRNAs) in A. oligospora remains scarcely described. Hereto, transcriptome sequencing was carried out to analyze the expression profiles of chitin-responsive miRNAs in A. oligospora. Based on the RNA-seq data, the differential expression of miRNAs (DEmiRNAs) in response to chitin was screened, identified and characterized in A. oligospora. Meanwhile, the potential target genes were predicted by the online tools miRanda and Targetscan, respectively. Furthermore, the interaction of DEmiRNA with it's target gene was validated by a dual-luciferase reporter assay system. Among 85 novel miRNAs identified, 25 miRNAs displayed significant differences in expression in A. oligospora in response to chitin. Gene Ontology (GO) analysis showed that the potential genes targeted by DEmiRNAs were enriched in the biological processes such as bio-degradation, extracellular components and cell cycle. KEGG analysis revealed that the target genes were mainly involved in Hippo, carbon and riboflavin metabolic pathway. Outstandingly, chitinase AOL_s00004g379, which is involved in the hydrolysis metabolic pathway of chitin, was confirmed to be a target gene of differential miR_70. These findings suggest that chitin-responsive miRNAs are involved in the regulation of cell proliferation, predator hyphae growth and chitinase expression through the mechanisms of post-transcriptional regulation, which provides a new perspective to the molecular mechanisms underlying miRNAs-mediated control of gene expression in A. oligospora.

Gene Cloning, Heterologous Expression, and In Silico Analysis of Chitinase B from Serratia marcescens for Biocontrol of Spodoptera frugiperda Larvae Infesting Maize Crops.

Spodoptera frugiperda, the fall armyworm (FAW), is a highly invasive polyphagous insect pest that is considered a source of severe economic losses to agricultural production. Currently, the majority of chemical insecticides pose tremendous threats to humans and animals besides insect resistance. Thus, there is an urgent need to develop new pest management strategies with more specificity, efficiency, and sustainability. Chitin-degrading enzymes, including chitinases, are promising agents which may contribute to FAW control. Chitinase-producing microorganisms are reported normally in bacteria and fungi. In the present study, Serratia marcescens was successfully isolated and identified from the larvae of Spodoptera frugiperda. The bacterial strain NRC408 displayed the highest chitinase enzyme activity of 250 units per milligram of protein. Subsequently, the chitinase gene was cloned and heterologously expressed in E. coli BL21 (DE3). Recombinant chitinase B was overproduced to 2.5-fold, driven by the T7 expression system. Recombinant chitinase B was evaluated for its efficacy as an insecticidal bioagent against S. frugiperda larvae, which induced significant alteration in subsequent developmental stages and conspicuous malformations. Additionally, our study highlights that in silico analyses of the anticipated protein encoded by the chitinase gene (ChiB) offered improved predictions for enzyme binding and catalytic activity. The effectiveness of (ChiB) against S. frugiperda was evaluated in laboratory and controlled field conditions. The results indicated significant mortality, disturbed development, different induced malformations, and a reduction in larval populations. Thus, the current study consequently recommends chitinase B for the first time to control FAW.

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.

Cloning of maize chitinase 1 gene and its expression in genetically transformed rice to confer resistance against rice blast caused by Pyricularia oryzae.

Fungal pathogens are one of the major reasons for biotic stress on rice (Oryza sativa L.), causing severe productivity losses every year. Breeding for host resistance is a mainstay of rice disease management, but conventional development of commercial resistant varieties is often slow. In contrast, the development of disease resistance by targeted genome manipulation has the potential to deliver resistant varieties more rapidly. The present study reports the first cloning of a synthetic maize chitinase 1 gene and its insertion in rice cv. (Basmati 385) via Agrobacterium-mediated transformation to confer resistance to the rice blast pathogen, Pyricularia oryzae. Several factors for transformation were optimized; we found that 4-week-old calli and an infection time of 15 minutes with Agrobacterium before colonization on co-cultivation media were the best-suited conditions. Moreover, 300 µM of acetosyringone in co-cultivation media for two days was exceptional in achieving the highest callus transformation frequency. Transgenic lines were analyzed using molecular and functional techniques. Successful integration of the gene into rice lines was confirmed by polymerase chain reaction with primer sets specific to chitinase and hpt genes. Furthermore, real-time PCR analysis of transformants indicated a strong association between transgene expression and elevated levels of resistance to rice blast. Functional validation of the integrated gene was performed by a detached leaf bioassay, which validated the efficacy of chitinase-mediated resistance in all transgenic Basmati 385 plants with variable levels of enhanced resistance against the P. oryzae. We concluded that overexpression of the maize chitinase 1 gene in Basmati 385 improved resistance against the pathogen. These findings will add new options to resistant germplasm resources for disease resistance breeding. The maize chitinase 1 gene demonstrated potential for genetic improvement of rice varieties against biotic stresses in future transformation programs.

Genome-Wide Identification and Expression Analysis of Chitinase Genes in Watermelon under Abiotic Stimuli and Fusarium oxysporum Infection.

Chitinases, which catalyze the hydrolysis of chitin, the primary components of fungal cell walls, play key roles in defense responses, symbiotic associations, plant growth, and stress tolerance. In this study, 23 chitinase genes were identified in watermelon (Citrullus lanatus [Thunb.]) and classified into five classes through homology search and phylogenetic analysis. The genes with similar exon-intron structures and conserved domains were clustered into the same class. The putative cis-elements involved in the responses to phytohormone, stress, and plant development were identified in their promoter regions. A tissue-specific expression analysis showed that the ClChi genes were primarily expressed in the roots (52.17%), leaves (26.09%), and flowers (34.78%). Moreover, qRT-PCR results indicate that ClChis play multifaceted roles in the interaction between plant/environment. More ClChi members were induced by Race 2 of Fusarium oxysporum f. sp. niveum, and eight genes were expressed at higher levels on the seventh day after inoculation with Races 1 and 2, suggesting that these genes play a key role in the resistance of watermelon to Fusarium wilt. Collectively, these results improve knowledge of the chitinase gene family in watermelon species and help to elucidate the roles played by chitinases in the responses of watermelon to various stresses.

Chitinous material bioconversion by three new chitinases from the yeast Mestchnikowia pulcherrima.

BACKGROUND: Chitinases are widely distributed enzymes that perform the biotransformation of chitin, one of the most abundant polysaccharides on the biosphere, into useful value-added chitooligosaccharides (COS) with a wide variety of biotechnological applications in food, health, and agricultural fields. One of the most important group of enzymes involved in the degradation of chitin comprises the glycoside hydrolase family 18 (GH18), which harbours endo- and exo-enzymes that act synergistically to depolymerize chitin. The secretion of a chitinase activity from the ubiquitous yeast Mestchnikowia pulcherrima and their involvement in the post-harvest biological control of fungal pathogens was previously reported. RESULTS: Three new chitinases from M. pulcherrima, MpChit35, MpChit38 and MpChit41, were molecularly characterized and extracellularly expressed in Pichia pastoris to about 91, 90 and 71 mU ml- 1, respectively. The three enzymes hydrolysed colloidal chitin with optimal activity at 45 ºC and pH 4.0-4.5, increased 2-times their activities using 1 mM of Mn2+ and hydrolysed different types of commercial chitosan. The partial separation and characterization of the complex COS mixtures produced from the hydrolysis of chitin and chitosan were achieved by a new anionic chromatography HPAEC-PAD method and mass spectrometry assays. An overview of the predicted structures of these proteins and their catalytic modes of action were also presented. Depicted their high sequence and structural homology, MpChit35 acted as an exo-chitinase producing di-acetyl-chitobiose from chitin while MpChit38 and MpChit41 both acted as endo-chitinases producing tri-acetyl-chitotriose as main final product. CONCLUSIONS: Three new chitinases from the yeast M. pulcherrima were molecularly characterized and their enzymatic and structural characteristics analysed. These enzymes transformed chitinous materials to fully and partially acetylated COS through different modes of splitting, which make them interesting biocatalysts for deeper structural-function studies on the challenging enzymatic conversion of chitin.

Functional Comparison of Three Chitinases from Symbiotic Bacteria of Entomopathogenic Nematodes.

Xenorhabdus and Photorhabdus, bacterial symbionts of entomopathogenic nematodes Steinernema and Heterorhabditis, respectively, have several biological activities including insecticidal and antimicrobial activities. Thus, XnChi, XhChi, and PtChi, chitinases of X. nematophila, X. hominickii, and P. temperata isolated from Korean indigenous EPNs S. carpocapsae GJ1-2, S. monticolum GJ11-1, and H. megidis GJ1-2 were cloned and expressed in Escherichia coli BL21 to compare their biological activities. Chitinase proteins of these bacterial symbionts purified using the Ni-NTA system showed different chitobiosidase and endochitinase activities, but N-acetylglucosamidinase activities were not shown in the measuring of chitinolytic activity through N-acetyl-D-glucosarmine oligomers. In addition, the proteins showed different insecticidal and antifungal activities. XnChi showed the highest insecticidal activity against Galleria mellonella, followed by PtChi and XhChi. In antifungal activity, XhChi showed the highest half-maximal inhibitory concentration (IC50) against Fusarium oxysporum with 0.031 mg/mL, followed by PtChi with 0.046 mg/mL, and XnChi with 0.072 mg/mL. XhChi also showed the highest IC50 against F. graminearum with 0.040 mg/mL, but XnChi was more toxic than PtChi with 0.055 mg/mL and 0.133 mg/mL, respectively. This study provides an innovative approach to the biological control of insect pests and fungal diseases of plants with the biological activity of symbiotic bacterial chitinases of entomopathogenic nematodes.

Functional importance of groups I and II chitinases, CHT5 and CHT10, in turnover of chitinous cuticle during embryo hatching and post-embryonic molting in the red flour beetle, Tribolium castaneum.

Chitinases (CHT) comprise a large gene family in insects and have been classified into at least eleven subgroups. Many studies involving RNA interference (RNAi) have demonstrated that depletion of group I (CHT5s) and group II (CHT10s) CHT transcripts causes lethal molting arrest in several insect species including the red flour beetle, Tribolium castaneum, presumably due to failure of degradation of chitin in their old cuticle. In this study we investigated the functions of CHT5 and CHT10 in turnover of chitinous cuticle in T. castaneum during embryonic and post-embryonic molting stages. RNAi and transmission electron microscopic (TEM) analyses indicate that CHT10 is required for cuticular chitin degradation at each molting period analyzed, while CHT5 is essential for pupal-adult molting only. We further analyzed the functions of these genes during embryogenesis in T. castaneum. Real-time qPCR analysis revealed that peak expression of CHT10 occurred prior to that of CHT5 during embryonic development as has been observed at post-embryonic molting periods in several other insect species. With immunogold-labeling TEM analysis using a fluorescein isothiocyanate-conjugated chitin-binding domain protein (FITC-CBD) probe, chitin was detected in the serosal cuticle but not in any other regions of the eggshell including the chorion and vitelline membrane layers. Injection of double-stranded RNA (dsRNA) for CHT5 (dsCHT5), CHT10 (dsCHT10) or their co-injection (dsCHT5/10) into mature adult females had no effect on their fecundity and the resulting embryos developed normally inside the egg. There were no obvious differences in the morphology of the outer chorion, inner chorion and vitelline membrane among eggs from these dsRNA-treated females. However, unlike dsCHT5 eggs, dsCHT10 and dsCHT5/10 eggs exhibited failure of turnover of the serosal cuticle in which the horizontal chitinous laminae remained intact, resulting in lethal embryo hatching defects. These results indicate that group I CHT5 is essential for pupal-adult molting, whereas group II CHT10 plays an essential role in cuticular chitin degradation in T. castaneum during both embryonic hatching and all of the post-embryonic molts. CHT10 can serve in place of CHT5 in chitin degradation, except during the pupal-adult molt when both enzymes are indispensable to complete eclosion.

Candida albicans chitinase 3 with potential as a vaccine antigen: production, purification, and characterisation.

Chitinases are widely studied enzymes that have already found widespread application. Their continued development and valorisation will be driven by the identification of new and improved variants and/or novel applications bringing benefits to industry and society. We previously identified a novel application for chitinases wherein the Candida albicans cell wall surface chitinase 3 (Cht3) was shown to have potential in vaccine applications as a subunit antigen against fungal infections. In the present study, this enzyme was investigated further, developing production and purification protocols, enriching our understanding of its properties, and advancing its application potential. Cht3 was heterologously expressed in Pichia pastoris and a 4-step purification protocol developed and optimised: this involves activated carbon treatment, hydrophobic interaction chromatography, ammonium sulphate precipitation, and gel filtration chromatography. The recombinant enzyme was shown to be mainly O-glycosylated and to retain the epitopes of the native protein. Functional studies showed it to be highly specific, displaying activity on chitin, chitosan, and chito-oligosaccharides larger than chitotriose only. Furthermore, it was shown to be a stable enzyme, exhibiting activity, and stability over broad pH and temperature ranges. This study represents an important step forward in our understanding of Cht3 and contributes to its development for application.

Development of a two-enzyme system in Aspergillus niger for efficient production of N-acetyl-ß-D-glucosamine from powdery chitin.

A chitinase (PbChi70) from Paenibacillus barengoltzii was engineered by directed evolution to enhance its hydrolysis efficiency towards powder chitin. Through two rounds of screening, a mutant (mPbChi70) with a maximum specific activity of 73.21 U/mg was obtained, which is by far the highest value ever reported. The mutant gene was further transformed into Aspergillus niger FBL-B (ΔglaA) which could secrete high level of endogenously ß-N-acetylglucosaminidase (GlcNAcase), thus a two-enzyme expression system was constructed. The highest chitinase activity of 61.33 U/mL with GlcNAcase activity of 353.1 U/mL was obtained in a 5-L fermentor by high-cell density fermentation. The chitin-degrading enzyme cocktail was used for the bioconversion of GlcNAc from powder chitin directly, and the highest conversion ratio reached high up to 71.9 % (w/w) with GlcNAc purity ≥95 % (w/w). This study may provide an excellent chitinase as well as a double enzyme cocktail system for efficient biological conversion of chitin materials.

The chitin-binding domain of Bacillus thuringiensis ChiA74 inhibits gram-negative bacterial and fungal pathogens of humans and plants.

The chitinase ChiA74 is synthesized by Bacillus thuringiensis and possesses a modular organization composed of four domains. In the C-terminal of the enzyme is located the chitin-binding domain (CBD), which has not been isolated as a single unit or characterized. Here, we aimed to isolate the ChiA74's CBD as a single unit, determine the binding properties, and evaluate its antimicrobial and hemolytic activities. We cloned the ChiA74's CBD and expressed it in Escherichia coli BL21. The single domain was purified, analyzed by SDS-PAGE, and characterized. The recombinant CBD (rCBD) showed a molecular mass of ∼14 kDa and binds strongly to α-chitin, with Kd and Bmax of ∼4.7 ± 0.9 µM and 1.5 ± 0.1 µmoles/g chitin, respectively. Besides, the binding potential (Bmax/Kd) was stronger for α-chitin (∼0.31) than microcrystalline cellulose (∼0.19). It was also shown that the purified rCBD inhibited the growth of the clinically relevant Gram-negative bacteria (GNB) Vibrio cholerae, and V. parahemolyticus CVP2 with minimum inhibitory concentrations (MICs) of 121 ± 9.9 and 138 ± 3.2 µg/mL, respectively, and of one of the most common GNB plant pathogens, Pseudomonas syringae with a MIC of 230 ± 13.8 µg/mL. In addition, the rCBD possessed antifungal activity inhibiting the conidia germination of Fusarium oxysporum (MIC = 192 ± 37.5 µg/mL) and lacked hemolytic and agglutination activities against human erythrocytes. The significance of this work lies in the fact that data provided here show for the first time that ChiA74's CBD from B. thuringiensis has antimicrobial activity, suggesting its potential use against significant pathogenic microorganisms. Future works will be focused on testing the inhibitory effect against other pathogenic microorganisms and elucidating the mechanism of action.

Identification of chitinase genes and roles in the larval-pupal transition of Leptinotarsa decemlineata.

BACKGROUND: Insect chitinases play crucial roles in degrading chitin in the extracellular matrix, affecting insect development and molting. However, our understanding of the specific functions of various chitinases in Leptinotarsa decemlineata is limited, hindering the deployment of novel gene-targeting technologies as pest management strategies. RESULTS: We identified and characterized 19 full-length complementary DNA (cDNA) sequences of chitinase genes (LdChts) in Leptinotarsa decemlineata. Despite having varying domain architectures, all these chitinases contained at least one chitinase catalytic domain. Phylogenetic analysis classified the chitinase proteins into ten distinct clusters (groups I-X). Expression profiles showed the highest expression in chitin-rich tissues or during specific developmental stages from the larva-to-pupa transition. Gene-specific RNA interference (RNAi) experiments provided valuable insight into chitinase gene function. Silencing of group II LdCht10 prevented larval-larval molting, larval-prepupal, and prepupal-pupal processes. Moreover, our study revealed that LdCht5, LdCht2, LdCht11, LdCht1, and LdCht3 from groups I and VII-X were specifically essential for the transition from prepupal to pupal stage, whereas LdIDGF2 from group V was necessary for the larval-prepupal metamorphic process. The chitinase gene LdCht7 from group III and LdIDGF4 from group V were involved in both the larva-to-prepupa and the prepupa-to-pupa shift. Additionally, our findings also shed light on the exclusive expression of nine chitinase genes within group IV in the digestive system, suggesting their potential role in regulating larval body weight and larva-to-pupa transition. CONCLUSION: Our results provide a comprehensive understanding of the functional specialization of chitinase genes during the molting process of various stages and identify potential targets for RNAi-based management of Leptinotarsa decemlineata. © 2023 Society of Chemical Industry.

The gastric mucosa of Atlantic salmon (Salmo salar) is abundant in highly active chitinases.

Atlantic salmon (Salmo salar) possesses a genome containing 10 genes encoding chitinases, yet their functional roles remain poorly understood. In other fish species, chitinases have been primarily linked to digestion, but also to other functions, as chitinase-encoding genes are transcribed in a variety of non-digestive organs. In this study, we investigated the properties of two chitinases belonging to the family 18 glycoside hydrolase group, namely Chia.3 and Chia.4, both isolated from the stomach mucosa. Chia.3 and Chia.4, exhibiting 95% sequence identity, proved inseparable using conventional chromatographic methods, necessitating their purification as a chitinase pair. Biochemical analysis revealed sustained chitinolytic activity against ß-chitin for up to 24 h, spanning a pH range of 2 to 6. Moreover, subsequent in vitro investigations established that this chitinase pair efficiently degrades diverse chitin-containing substrates into chitobiose, highlighting the potential of Atlantic salmon to utilize novel chitin-containing feed sources. Analysis of the gastric matrix proteome demonstrates that the chitinases are secreted and rank among the most abundant proteins in the gastric matrix. This finding correlates well with the previously observed high transcription of the corresponding chitinase genes in Atlantic salmon stomach tissue. By shedding light on the secreted chitinases in the Atlantic salmon's stomach mucosa and elucidating their functional characteristics, this study enhances our understanding of chitinase biology in this species. Moreover, the observed capacity to effectively degrade chitin-containing materials implies the potential utilization of alternative feed sources rich in chitin, offering promising prospects for sustainable aquaculture practices.

Polysaccharide degradation in Cellvibrionaceae: Genomic insights of the novel chitin-degrading marine bacterium, strain KSP-S5-2, and its chitinolytic activity.

In this study, we present the genome characterization of a novel chitin-degrading strain, KSP-S5-2, and comparative genomics of 33 strains of Cellvibrionaceae. Strain KSP-S5-2 was isolated from mangrove sediment collected in Balik Pulau, Penang, Malaysia, and its 16S rRNA gene sequence showed the highest similarity (95.09%) to Teredinibacter franksiae. Genome-wide analyses including 16S rRNA gene sequence similarity, average nucleotide identity, digital DNA-DNA hybridization, and phylogenomics, suggested that KSP-S5-2 represents a novel species in the family Cellvibrionaceae. The Cellvibrionaceae pan-genome exhibited high genomic variability, with only 1.7% representing the core genome, while the flexible genome showed a notable enrichment of genes related to carbohydrate metabolism and transport pathway. This observation sheds light on the genetic plasticity of the Cellvibrionaceae family and the gene pools that form the basis for the evolution of polysaccharide-degrading capabilities. Comparative analysis of the carbohydrate-active enzymes across Cellvibrionaceae strains revealed that the chitinolytic system is not universally present within the family, as only 18 of the 33 genomes encoded chitinases. Strain KSP-S5-2 displayed an expanded repertoire of chitinolytic enzymes (25 GH18, two GH19 chitinases, and five GH20 ß-N-acetylhexosaminidases) but lacked genes for agar, xylan, and pectin degradation, indicating specialized enzymatic machinery focused primarily on chitin degradation. Further, the strain degraded 90% of chitin after 10 days of incubation. In summary, our findings provided insights into strain KSP-S5-2's genomic potential, the genetics of its chitinolytic system, genomic diversity within the Cellvibrionaceae family in terms of polysaccharide degradation, and its application for chitin degradation.

Engineering the Relatively Conserved Residues in Active Site Architecture of Thermophilic Chitinase SsChi18A Enhanced Catalytic Activity.

Chitinase plays a vital role in the efficient biotransformation of the chitin substrate. This study aimed to modify and elucidate the contribution of the relatively conserved residues in the active site architecture of a thermophilic chitinase SsChi18A from Streptomyces sp. F-3 in processive catalysis. The enzymatic activity on colloidal chitin increased to 151%, 135%, and 129% in variants Y286W, E287A, and K186A compared with the wild type (WT). Also, the apparent processive parameter G2/G1 was lower in the variants compared to the WT, indicating the essential role of Tyr-286, Glu-287, and Lys-186 in processive catalysis. Additionally, the enzymatic activity on the crystalline chitin of F48W and double mutants F48W/Y209F and F48W/Y286W increased by 35%, 16%, and 36% compared with that for WT. Molecular dynamics simulations revealed that the driving force of processive catalysis might be related to the changes in interaction energy. This study provided a rational design strategy targeting relatively conserved residues to enhance the catalytic activity of GH18 processive chitinases.

An overview of viral chitinases: General properties and biotechnological potential.

Chitin is a biopolymer profusely present in nature and of pivotal importance as a structural component in cells. It is degraded by chitinases, enzymes naturally produced by different organisms. Chitinases are proteins enrolled in many cellular mechanisms, including the remodeling process of the fungal cell wall, the cell growth process, the autolysis of filamentous fungi, and cell separation of yeasts, among others. These enzymes also have properties with different biotechnological applications. They are used to produce polymers, for biological control, biofilm formation, and as antitumor and anti-inflammatory target molecules. Chitinases are classified into different glycoside hydrolase (GH) families and are widespread in microorganisms, including viruses. Among them, the GH18 family is highly predominant in the viral genomes, being present and active enzymes in baculoviruses and nucleocytoplasmic large DNA viruses (NCLDV), especially chloroviruses from the Phycodnaviridae family. These viral enzymes contain one or more GH domains and seem to be involved during the viral replication cycle. Curiously, only a few DNA viruses have these enzymes, and studying their properties could be a key feature for biological and biotechnological novelties. Here, we provide an overview of viral chitinases and their probable function in viral infection, showing evidence of at least two distinct origins for these enzymes. Finally, we discuss how these enzymes can be applied as biotechnological tools and what one can expect for the coming years on these GHs.

Dibenzylideneacetone Overcomes Botrytis cinerea Infection in Cherry Tomatoes by Inhibiting Chitinase Activity.

Chitinase, a crucial component of the fungal cell wall and septa, plays an important role in fungal germination by hydrolyzing chitin to provide carbon and energy for fungal growth and reproduction. In this study, we initially screened dibenzylideneacetone (DBA), a small molecule with inhibitory activity against Botrytis cinerea Chitinase, exhibiting an IC50 of 13.10 µg/mL. By constructing a three-dimensional (3D) model of the B. cinerea Chitinase and utilizing computational biology approaches, we found DBA bound to the active site pocket and formed strong π-π interactions and hydrophobic interactions with Chitinase, indicative of its competitive inhibitory mode. Site-directed mutagenesis also revealed that TRP-382, TRP-135, and ALA-215 were key amino acid residues involved in DBA binding. Subsequent antifungal assays showed that DBA had an MIC of 32 µg/mL against B. cinerea and EC50 values of 16.29 and 14.64 µg/mL in inhibiting mycelial growth and spore germination, respectively. Importantly, in vivo experiments demonstrated that DBA treatment significantly extended the shelf life of cherry tomatoes by 2-fold. Therefore, DBA represents a promising antifungal agent for fruit preservation applications.