Chitin-modifying Enzymes Research Articles

ENDOPHYTIC FUNGI AS CHITIN-MODIFYING ENZYMES PRODUCERS

Chitosan and chitooligosaccharides, which are products of chitin modification, have wide industrial applications. An improved understanding of the structure and properties of chitin-modifying enzymes has increased the interest in obtaining them from microbial sources and using them in the biotechnological production of these essential sugars. Endophytic fungi include a diverse group of microorganisms that inhabit the intercellular and intracellular areas of plant tissues, thus exerting beneficial effects on host species. They are considered an extremely valuable source of biologically active secondary metabolites and enzymes with high application potential. This review presents the potential of endophytic fungi to produce chitin-modifying enzymes and discusses the role of chitin modification by enzymes produced by fungal endophytes in their survival in the plant host.

The cuticle proteome of a planktonic crustacean.

The cuticles of arthropods provide an interface between the organism and its environment. Thus, the cuticle's structure influences how the organism responds to and interacts with its surroundings. Here, we used label-free quantification proteomics to provide a proteome of the moulted cuticle of the aquatic crustacean Daphnia magna, which has long been a prominent subject of studies on ecology, evolution, and developmental biology. We detected a total of 278 high-confidence proteins. Using protein sequence domain and functional enrichment analyses, we identified chitin-binding structural proteins and chitin-modifying enzymes as the most abundant protein groups in the cuticle proteome. Structural cuticular protein families showed a similar distribution to those found in other arthropods and indicated proteins responsible for the soft and flexible structure of the Daphnia cuticle. Finally, cuticle protein genes were also clustered as tandem gene arrays in the D. magna genome. The cuticle proteome presented here will be a valuable resource to the Daphnia research community, informing genome annotations and investigations on diverse topics such as the genetic basis of interactions with predators and parasites.

Role of Chitin Deacetylase 1 in the Molting and Metamorphosis of the Cigarette Beetle Lasioderma serricorne

Chitin deacetylases (CDAs) are chitin-modifying enzymes known to play vital roles in insect metamorphosis and development. In this study, we identified and characterized a chitin deacetylase 1 gene (LsCDA1) from the cigarette beetle Lasioderma serricorne. LsCDA1 contains a 1614 bp open reading frame encoding a protein of 537 amino acids that includes domain structures typical of CDAs. LsCDA1 was mainly expressed in the late larval and late pupal stages. In larval tissues, the highest level of LsCDA1 was detected in the integument. The expression of LsCDA1 was induced by 20-hydroxyecdysone (20E) in vivo, and it was significantly suppressed by knocking down the expression of ecdysteroidogenesis genes and 20E signaling genes. RNA interference (RNAi)-aided silencing of LsCDA1 in fifth-instar larvae prevented the larval–pupal molt and caused 75% larval mortality. In the late pupal stage, depletion of LsCDA1 resulted in the inhibition of pupal growth and wing abnormalities, and the expression levels of four wing development-related genes (LsDY, LsWG, LsVG, and LsAP) were dramatically decreased. Meanwhile, the chitin contents of LsCDA1 RNAi beetles were significantly reduced, and expressions of three chitin synthesis pathway genes (LsTRE1, LsUAP1, and LsCHS1) were greatly decreased. The results suggest that LsCDA1 is indispensable for larval–pupal and pupal–adult molts, and that it is a potential target for the RNAi-based control of L. serricorne.

Biochemical characterization of three midgut chitin deacetylases of the Lepidopteran insect Bombyx mori

Peritrophic membrane (PM) is a chitin and protein-containing extracellular matrix that lines the midgut in most insect species, functioning as a barrier to exogenous toxins and pathogens. Midgut chitin deacetylases (CDAs) are chitin-modifying enzymes known to alter the mechanical property and permeability of PM. However, biochemical properties and specific roles of these enzymes remain elusive. In this study, the midgut-expressed CDAs (BmCDA6, BmCDA7 and BmCDA8) from Bombyx mori were cloned, recombinantly expressed and purified and their enzymatic activities toward PM chitin were determined. Of the three enzymes, BmCDA7 exhibited the highest activity (0.284 μmol/min/μmol), while BmCDA8 showed lower activity of 0.061 μmol/min/μmol. BmCDA6 was inactive towards PM chitin. Gene expression patterns indicated that although all three CDA genes were specifically expressed in the anterior midgut, they differed in their temporal expression patterns. BmCDA6 was expressed almost exclusively at the mid-molt stage, the stage when the PM was thick and with multiple chitin layers. Unlike BmCDA6, high expression levels of BmCDA7 and BmCDA8 were observed only at the feeding stage, the stage when the PM is thin and with fewer chitin layers. The different gene expression patterns and biochemical characteristics provide new information about the functional specialization among BmCDA6, BmCDA7 and BmCDA8 proteins.

Endochitinase 1 (Tv-ECH1) from Trichoderma virens has high subsite specificities for acetylated units when acting on chitosans

Chitosans with defined characteristics have been shown to possess reproducible bioactivities for numerous applications. A promising approach for producing chitosans with defined degrees of polymerization (DP), degrees of acetylation (DA), and patterns of acetylation (PA) involves using chitin-modifying enzymes. One such enzyme, the chitinase Tv-ECH1 belonging to the glycoside hydrolase (GH) family 18, seems to have an important role in the biocontrol properties of the fungus Trichoderma virens, suggesting its potential in generating novel chitosans for plant health applications. In this study, the Tv-ECH1 enzyme was overexpressed in the methylotrophic yeast Pichia pastoris, yielding large amounts (up to 2mgmL−1) of purified recombinant enzyme of high activity, high purity, and high stability, making the system promising for industrial production of Tv-ECH1. The purified Tv-ECH1 chitinase displayed a wide optimal pH range from 4.5 to 6 and an optimal temperature of 37°C. Detailed subsite specificity analyses revealed high preference for acetylated residues at all four subsites analyzed (−2, −1, +1, +2), making Tv-ECH1 a promising candidate for the biotechnological production of specific chitosan oligomers and for the characterization of chitosan polymers via enzymatic fingerprinting.

Immune function against bacteria of chitin deacetylase 1 (EcCDA1) from Exopalaemon carinicauda

Chitin deacetylase (CDA, EC 3.5.1.41), belonging to a family of extracellular chitin-modifying enzymes, can catalyze the deacetylation of chitin. In this study, the full-length cDNA sequence encoding chitin deacetylase 1 (EcCDA1) was obtained fromExopalaemon carinicauda. The complete nucleotide sequence of EcCDA1 contained a 1611 bp open reading frame (ORF) encoding EcCDA1 precursor of 536 amino acids. The domain architecture of the deduced EcCDA1 protein contained a signal peptide, a chitin-binding peritrophin-A domain (ChtBD2), a low-density lipoprotein receptor class A domain (LDLa) and a Polysacc_deac_1 domain. EcCDA1 mRNA was predominantly expressed in the gills. The expression of EcCDA1 in the prawns challenged with Vibrio parahaemolyticus and Aeromonas hydrophila changed in a time-dependent manner. The expression of EcCDA1 in the prawns challenged with V. parahaemolyticus was up-regulated at 12 h (p < 0.05), and significantly up-regulated at 24 h and 48 h (p < 0.01), and then returned to the control levels at 96 h post-challenge (p > 0.05). At the same time, the expression in Aeromonas-challenged group was significantly up-regulated at 12, 24 and 48 h (p < 0.01) and returned to the control levels at 120 h post-challenge (p > 0.05). Then, EcCDA1 was recombinantly expressed in Pichia pastoris and the purified recombinant EcCDA1 could not inhibit the growth of V. parahaemolyticus or A. hydrophila, which indicated that the CDA1 may play its biological activity in immune defense by deacetylation from chitin.

Rac1 controls epithelial tube length through the apical secretion and polarity pathways.

ABSTRACTThe morphometric parameters of epithelial tubes are critical to the physiology and homeostasis of most organs. In addition, many human diseases are associated with tube-size defects. Here, we show that Rac1 limits epithelial tube elongation in the developing fly trachea by promoting Rab5-dependent endocytosis of the apical determinant Crumbs. Rac1 is also involved in a positive feedback loop with the septate junction protein Coracle. Thereby, Rac1 precludes paracellular diffusion and contributes to the septate junction-dependent secretion of the chitin-modifying enzymes Vermiform and Serpentine, which restrict epithelial tube length independently of Crumbs. Thus, Rac1 is a critical component of two important pathways controlling epithelial tube morphogenesis.

Proteomic analysis of Daphnia magna hints at molecular pathways involved in defensive plastic responses.

BackgroundPhenotypic plasticity in defensive traits occurs in many species when facing heterogeneous predator regimes. The waterflea Daphnia is well-known for showing a variety of these so called inducible defences. However, molecular mechanisms underlying this plasticity are poorly understood so far. We performed proteomic analysis on Daphnia magna exposed to chemical cues of the predator Triops cancriformis. D. magna develops an array of morphological changes in the presence of Triops including changes of carapace morphology and cuticle hardening.ResultsUsing the 2D-DIGE technique, 1500 protein spots could be matched and quantified. We discovered 179 protein spots with altered intensity when comparing Triops exposed animals to a control group, and 69 spots were identified using nano-LC MS/MS. Kairomone exposure increased the intensity of spots containing muscle proteins, cuticle proteins and chitin-modifying enzymes as well as enzymes of carbohydrate and energy metabolism. The yolk precursor protein vitellogenin decreased in abundance in 41 of 43 spots.ConclusionIdentified proteins may be either directly involved in carapace stability or reflect changes in energy demand and allocation costs in animals exposed to predator kairomones. Our results present promising candidate proteins involved in the expression of inducible defences in Daphnia and enable further in depth analysis of this phenomenon.

Chitinolytic enzymes from endophytic fungi

Fungal endophytes isolated from leaves of tree species of the forests of Western Ghats, southern India were screened for chitin modifying enzyme production. Thirty-one of the one hundred and sixty two isolates were positive for chitinase, while different isolates produced isoforms of the enzyme. Many isolates produced chitosanase that acted on chitosan with different degrees of acetylation. Modified chitin and different types of chitosans are used in biomedical applications including wound healing, drug delivery, gene delivery, tissue engineering, in the food industry as preservatives and emulsifying agents, and in biocatalysis. Horizontally transmitted endophytes appear to be a good source for a variety of chitin modifying enzymes with the potential to be used in biotechnology. The possibility of chitin modifying enzymes of endophytes in regulating plant defense against pathogens and pests in vivo should also be addressed.

Domain organization and phylogenetic analysis of proteins from the chitin deacetylase gene family of Tribolium castaneum and three other species of insects

A bioinformatics investigation of four insect species with annotated genome sequences identified a family of genes encoding chitin deacetylase (CDA)-like proteins, with five to nine members depending on the species. CDAs (EC 3.5.1.41) are chitin-modifying enzymes that deacetylate the β-1,4-linked N-acetylglucosamine homopolymer. Partial deacetylation forms a heteropolysaccharide that also contains some glucosamine residues, while complete deacetylation produces the homopolymer chitosan, consisting exclusively of glucosamine. The genomes of the red flour beetle, Tribolium castaneum, the fruit fly, Drosophila melanogaster, the malaria mosquito, Anopheles gambiae, and the honey bee, Apis mellifera contain 9, 6, 5 and 5 genes, respectively, that encode proteins with a chitin deacetylase motif. The presence of alternative exons in two of the genes, TcCDA2 and TcCDA5, increases the protein diversity further. Insect CDA-like proteins were classified into five orthologous groups based on phylogenetic analysis and the presence of additional motifs. Group I enzymes include CDA1 and isoforms of CDA2, each containing in addition to a polysaccharide deacetylase-like catalytic domain, a chitin-binding peritrophin-A domain (ChBD) and a low-density lipoprotein receptor class A domain (LDLa). Group II is composed of CDA3 orthologs from each insect species with the same domain organization as group I CDAs, but differing substantially in sequence. Group III includes CDA4s, which have the ChBD domain but do not have the LDLa domain. Group IV comprises CDA5s, which are the largest CDAs because of a very long intervening region separating the ChBD and catalytic domains. Among the four insect species, Tribolium is unique in having four CDA genes in group V, whereas the other insect genomes have either one or none. Most of the CDA-like proteins have a putative signal peptide consistent with their role in modifying extracellular chitin in both cuticle and peritrophic membrane during morphogenesis and molting.

Detection and characterization of chitinases and other chitin-modifying enzymes.

Multiple industrial and medical uses of chitin and its derivatives have been developed in recent years. The demand for enzymes with new or desirable properties continues to grow as additional uses of chitin, chitooligosaccharides, and chitosan become apparent. Microorganisms, the primary degraders of chitin in the environment, are a rich source of valuable chitin-modifying enzymes. This review summarizes many methods that can be used to isolate and characterize chitin-modifying enzymes including chitin depolymerases, chitodextrinases, chitin deacetylases, N-acetylglucosaminidases, chitin-binding proteins, and chitosanases. Chitin analogs, zymography, detection of reducing sugars, genomic library screening, chitooligosaccharide electrophoresis, degenerate PCR primer design, thin layer chromatography, and chitin-binding assays are discussed.