scispace - formally typeset
Search or ask a question

Showing papers on "Chitinase published in 2014"


Journal ArticleDOI
TL;DR: Other emerging technologies such as direct degradation of chitin from crustacean shells and microbial cell walls, enzymatic synthesis of COS from small building blocks, and protein engineering technology for chit in-related enzymes have been discussed as the most significant challenge for industrial application.
Abstract: Chitin and chitosan oligosaccharides (COS) have been traditionally obtained by chemical digestion with strong acids. In light of the difficulties associated with these traditional production processes, environmentally compatible and reproducible production alternatives are desirable. Unlike chemical digestion, biodegradation of chitin and chitosan by enzymes or microorganisms does not require the use of toxic chemicals or excessive amounts of wastewater. Enzyme preparations with chitinase, chitosanase, and lysozymeare primarily used to hydrolyze chitin and chitosan. Commercial preparations of cellulase, protease, lipase, and pepsin provide another opportunity for oligosaccharide production. In addition to their hydrolytic activities, the transglycosylation activity of chitinolytic enzymes might be exploited for the synthesis of desired chitin oligomers and their derivatives. Chitin deacetylase is also potentially useful for the preparation of oligosaccharides. Recently, direct production of oligosaccharides from chitin and crab shells by a combination of mechanochemical grinding and enzymatic hydrolysis has been reported. Together with these, other emerging technologies such as direct degradation of chitin from crustacean shells and microbial cell walls, enzymatic synthesis of COS from small building blocks, and protein engineering technology for chitin-related enzymes have been discussed as the most significant challenge for industrial application.

167 citations


Journal ArticleDOI
N. Ashwini1, S. Srividya1
01 Apr 2014
TL;DR: A soil bacterium, Bacillus subtilis, isolated from the rhizosphere of Chilli, showed high antagonistic activity against Colletotrichum gloeosporioides OGC1 and produced other mycolytic enzymes—glucanase and cellulase, demonstrated by a clear zone of hydrolysis of yeast cell wall glucan and carboxymethylcellulose.
Abstract: A soil bacterium, Bacillus subtilis, isolated from the rhizosphere of Chilli, showed high antagonistic activity against Colletotrichum gloeosporioides OGC1. A clear inhibition zone of 0.5–1 cm was observed in dual plate assay. Microscopic observations showed a clear hyphal lysis and degradation of fungal cell wall. In dual liquid cultures, the B. subtilis strain inhibited the C. gloeosporioides up to 100 % in terms of dry weight. This strain also produced a clear halo region on chitin agar medium plates containing 0.5 % colloidal chitin, indicating that it excretes chitinase. The strain also produced other mycolytic enzymes—glucanase and cellulase, demonstrated by a clear zone of hydrolysis of yeast cell wall glucan (YCW 0.1 % v/v) and carboxymethylcellulose (CMC 0.1 % v/v). In liquid cultures, the strain showed appreciable levels of chitinase, glucanase and cellulase activities and hydrolytic activity with C. gloeosporioides OGC1 mycelia as the substrate. The role of the B. subtilis strain in suppressing the fungal growth in vitro was studied in comparison with a UV mutant of that strain, which lacked both antagonistic and hydrolytic activity. The mycolytic enzyme mediated antagonism of B. subtilis was further demonstrated by heat inactivation (70–100 °C), treatment with trypsin and TCA of the crude enzyme extract which lacked antifungal property also. Treatment of the chilli seeds with Bacillus sp. culture showed 100 % germination index similar to the untreated seeds. The treatment of the seed with co-inoculation of the pathogen with Bacillus sp. culture showed 65 % reduction in disease incidence by the treatment as compared to the seed treated with pathogen alone (77.5 %).

133 citations


Journal ArticleDOI
02 Sep 2014-eLife
TL;DR: It is found that chitinase is essential for bacteria to enter hyphae and grant unprecedented insight into the fungal cell wall penetration and symbiosis formation.
Abstract: The rice seedling blight fungus Rhizopus microsporus and its endosymbiont Burkholderia rhizoxinica form an unusual, highly specific alliance to produce the highly potent antimitotic phytotoxin rhizoxin. Yet, it has remained a riddle how bacteria invade the fungal cells. Genome mining for potential symbiosis factors and functional analyses revealed that a type 2 secretion system (T2SS) of the bacterial endosymbiont is required for the formation of the endosymbiosis. Comparative proteome analyses show that the T2SS releases chitinolytic enzymes (chitinase, chitosanase) and chitin-binding proteins. The genes responsible for chitinolytic proteins and T2SS components are highly expressed during infection. Through targeted gene knock-outs, sporulation assays and microscopic investigations we found that chitinase is essential for bacteria to enter hyphae. Unprecedented snapshots of the traceless bacterial intrusion were obtained using cryo-electron microscopy. Beyond unveiling the pivotal role of chitinolytic enzymes in the active invasion of a fungus by bacteria, these findings grant unprecedented insight into the fungal cell wall penetration and symbiosis formation.

106 citations


Journal ArticleDOI
TL;DR: This review covers the recent advances of chitinases as a biocontrol agent and its various applications including preparation of medically important chitooligosaccharides, bioconversion of Chitin as well as in implementing chit inases as diagnostic and prognostic markers for numerous diseases and the prospect of their future utilization.
Abstract: Biological control of phytopathogenic fungi and insects continues to inspire the research and development of environmentally friendly bioactive alternatives. Potentially lytic enzymes, chitinases can act as a biocontrol agent against agriculturally important fungi and insects. The cell wall in fungi and protective covers, i.e. cuticle in insects shares a key structural polymer, chitin, a β-1,4-linked N-acetylglucosamine polymer. Therefore, it is advantageous to develop a common biocontrol agent against both of these groups. As chitin is absent in plants and mammals, targeting its metabolism will signify an eco-friendly strategy for the control of agriculturally important fungi and insects but is innocuous to mammals, plants, beneficial insects and other organisms. In addition, development of chitinase transgenic plant varieties probably holds the most promising method for augmenting agricultural crop protection and productivity, when properly integrated into traditional systems. Recently, human proteins with chitinase activity and chitinase-like proteins were identified and established as biomarkers for human diseases. This review covers the recent advances of chitinases as a biocontrol agent and its various applications including preparation of medically important chitooligosaccharides, bioconversion of chitin as well as in implementing chitinases as diagnostic and prognostic markers for numerous diseases and the prospect of their future utilization.

96 citations


Journal ArticleDOI
TL;DR: Results indicate that ChiA is a soluble extracellular chit inase required for chitin utilization and that it relies on a novel CTD for secretion by the F. johnsoniae T9SS.
Abstract: Flavobacterium johnsoniae, a member of phylum Bacteriodetes, is a gliding bacterium that digests insoluble chitin and many other polysaccharides. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding motility and for chitin utilization. Five potential chitinases were identified by genome analysis. Fjoh_4555 (ChiA), a 168.9-kDa protein with two glycoside hydrolase family 18 (GH18) domains, was targeted for analysis. Disruption of chiA by insertional mutagenesis resulted in cells that failed to digest chitin, and complementation with wild-type chiA on a plasmid restored chitin utilization. Antiserum raised against recombinant ChiA was used to detect the protein and to characterize its secretion by F. johnsoniae. ChiA was secreted in soluble form by wild-type cells but remained cell associated in strains carrying mutations in any of the T9SS genes, gldK, gldL, gldM, gldNO, sprA, sprE, and sprT. Western blot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses suggested that ChiA was proteolytically processed into two GH18 domain-containing proteins. Proteins secreted by T9SSs typically have conserved carboxy-terminal domains (CTDs) belonging to the TIGRFAM families TIGR04131 and TIGR04183. ChiA does not exhibit strong similarity to these sequences and instead has a novel CTD. Deletion of this CTD resulted in accumulation of ChiA inside cells. Fusion of the ChiA CTD to recombinant mCherry resulted in secretion of mCherry into the medium. The results indicate that ChiA is a soluble extracellular chitinase required for chitin utilization and that it relies on a novel CTD for secretion by the F. johnsoniae T9SS.

85 citations


Journal ArticleDOI
TL;DR: Chi18H8 is the first chitinase isolated from a metagenome library obtained in pure form and which has the potential to be used as a candidate agent for controlling fungal crop diseases and may also answer to the demand for novel chit in-degrading enzymes for a broad range of other industrial processes and medical purposes.
Abstract: Plant disease caused by fungal pathogens results in vast crop damage globally. Microbial communities of soil that is suppressive to fungal crop disease provide a source for the identification of novel enzymes functioning as bioshields against plant pathogens. In this study, we targeted chitin-degrading enzymes of the uncultured bacterial community through a functional metagenomics approach, using a fosmid library of a suppressive soil metagenome. We identified a novel bacterial chitinase, Chi18H8, with antifungal activity against several important crop pathogens. Sequence analyses show that the chi18H8 gene encodes a 425-amino acid protein of 46 kDa with an N-terminal signal peptide, a catalytic domain with the conserved active site F175DGIDIDWE183, and a chitinase insertion domain. Chi18H8 was expressed (pGEX-6P-3 vector) in Escherichia coli and purified. Enzyme characterization shows that Chi18H8 has a prevalent chitobiosidase activity with a maximum activity at 35 °C at pH lower than 6, suggesting a role as exochitinase on native chitin. To our knowledge, Chi18H8 is the first chitinase isolated from a metagenome library obtained in pure form and which has the potential to be used as a candidate agent for controlling fungal crop diseases. Furthermore, Chi18H8 may also answer to the demand for novel chitin-degrading enzymes for a broad range of other industrial processes and medical purposes.

76 citations


Journal ArticleDOI
TL;DR: The excellent thermostability and hydrolytic properties may give the exochitinase great potential in GlcNAc production from chitin, the first report on an exoch itinase with N-acetyl-β-D-glucosaminidase activity from Paenibacillus species.
Abstract: Background N-acetyl-β-D-glucosamine (GlcNAc) is widely used as a valuable pharmacological agent and a functional food additive. The traditional chemical process for GlcNAc production has some problems such as high production cost, low yield, and acidic pollution. Hence, to identify a novel chitinase that is suitable for bioconversion of chitin to GlcNAc is of great value.

68 citations


Journal ArticleDOI
TL;DR: It is demonstrated that specific enzymes ChtI, ChtII, Chi-h, and Hex1 can be assigned to cuticular chitin degradation, which is extremely important for insect growth and development.
Abstract: Cuticular chitin degradation is extremely important for insect growth and development, which has not been fully understood thus far. One obstacle to understanding this mechanism is the lack of a systematic analysis of the chitinolytic enzymes involved in cuticular chitin degradation. In this study, we used the silkmoth Bombyx mori as a model organism and compared proteomic analyses for larval-pupal (L-P) and pupal-adult (P-A) molting fluids using tandem mass tag quantitative mass spectrometry. There were 195 proteins identified from both L-P and P-A molting fluids. A total of 170 out of 195 proteins were deduced to be secretory and were enriched for GO terms associated with chitin metabolism and proteolysis by using AgriGO. Although the chitinolytic enzymes are encoded by many insect genes, the proteomics analysis unexpectedly showed that only four chitinolytic enzymes with the combination "211" were abundant in both molting fluids, namely, two insect GH18 Chitinase family members (ChtI and ChtII), one bacterial-type GH18 Chitinase (Chi-h), and one insect GH20 hexosaminidase (Hex1). A tissue-specific and stage-specific gene expression pattern verified that the "211" enzymes are involved in cuticular chitin degradation. This work first demonstrates that specific enzymes ChtI, ChtII, Chi-h, and Hex1 can be assigned to cuticular chitin degradation.

68 citations


Journal ArticleDOI
TL;DR: The developed ChitO-based assay can detect chitinase activity as low as 10 μU within 15 minutes of assay time and cellulase activity can be detected in the range of 6 to 375 mU, which renders this newly developed assay method highly suitable for applications in biorefinery-related research.
Abstract: Most of the current colorimetric methods for detection of chitinase or cellulase activities on the insoluble natural polymers chitin and cellulose depend on a chemical redox reaction. The reaction involves the reducing ends of the hydrolytic products. The Schales’ procedure and the 3,5-dinitrosalicylic acid (DNS) method are two examples that are commonly used. However, these methods lack sensitivity and present practical difficulties of usage in high-throughput screening assays as they require boiling or heating steps for color development. We report a novel method for colorimetric detection of chitinase and cellulase activity. The assay is based on the use of two oxidases: wild-type chito-oligosaccharide oxidase, ChitO, and a mutant thereof, ChitO-Q268R. ChitO was used for chitinase, while ChitO-Q268R was used for cellulase activity detection. These oxidases release hydrogen peroxide upon the oxidation of chitinase- or cellulase-produced hydrolytic products. The hydrogen peroxide produced can be monitored using a second enzyme, horseradish peroxidase (HRP), and a chromogenic peroxidase substrate. The developed ChitO-based assay can detect chitinase activity as low as 10 μU within 15 minutes of assay time. Similarly, cellulase activity can be detected in the range of 6 to 375 mU. A linear response was observed when applying the ChitO-based assay for detecting individual chito-oligosaccharides and cello-oligosaccharides. The detection limits for these compounds ranged from 5 to 25 μM. In contrast to the other commonly used methods, the Schales’ procedure and the DNS method, no boiling or heating is needed in the ChitO-based assays. The method was also evaluated for detecting hydrolytic activity on biomass-derived substrates, that is, wheat straw as a source of cellulose and shrimp shells as a source of chitin. The ChitO-based assay has clear advantages for the detection of chitinase and cellulase activity over the conventional Schales’ procedure and DNS method. The detection limit is lower and there is no requirement for harsh conditions for the development of the signal. The assay also involves fewer and easier handling steps. There is no need for boiling to develop the color and results are available within 15 minutes. These aforementioned features render this newly developed assay method highly suitable for applications in biorefinery-related research.

64 citations


Journal ArticleDOI
TL;DR: Positive responses of ScChi to the biotic and abiotic stimuli reveal that this gene is a stress-related gene of sugarcane, suggesting a close relationship between the expression of Sc Chi and plant immunity.
Abstract: Chitinases (EC 3.2.2.14), expressed during the plant-pathogen interaction, are associated with plant defense against pathogens. In the present study, a positive correlation between chitinase activity and sugarcane smut resistance was found. ScChi (GenBank accession no. KF664180), a Class III chitinase gene, encoded a 31.37 kDa polypeptide, was cloned and identified. Subcellular localization revealed ScChi targeting to the nucleus, cytoplasm and the plasma membrane. Real-time quantitative PCR (RT-qPCR) results showed that ScChi was highly expressed in leaf and stem epidermal tissues. The ScChi transcript was both higher and maintained longer in the resistance cultivar during challenge with Sporisorium scitamineum. The ScChi also showed an obvious induction of transcription after treatment with SA (salicylic acid), H2O2, MeJA (methyl jasmonate), ABA (abscisic acid), NaCl, CuCl2, PEG (polyethylene glycol) and low temperature (4 °C). The expression levels of ScChi and six immunity associated marker genes were upregulated by the transient overexpression of ScChi. Besides, histochemical assay of Nicotiana benthamiana leaves overexpressing pCAMBIA 1301-ScChi exhibited deep DAB (3,3'-diaminobenzidinesolution) staining color and high conductivity, indicating the high level of H2O2 accumulation. These results suggest a close relationship between the expression of ScChi and plant immunity. In conclusion, the positive responses of ScChi to the biotic and abiotic stimuli reveal that this gene is a stress-related gene of sugarcane.

60 citations


Journal ArticleDOI
TL;DR: Chitinase enzyme purified from Bacillus subtilis TV-125A was tested on four fungal agents, and although all the results were positive, it was particularly effective on F. culmorum according to the findings.
Abstract: Chitin is the main structural component of cell walls of fungi, exoskeletons of insects and other arthropods and shells of crustaceans. Chitinase enzyme is capable of degrading chitin, and this enzyme can be used as a biological fungicide against phytopathogenic fungi, as well as an insecticide against insect pests. In this study, 158 isolates, which were derived from bacteria cultures isolated from leaves and root rhizospheres of certain plants in Turkey, were selected after confirming that they are not phytopathogenic based on the hypersensitivity test performed on tobacco; and antifungal activity test was performed against Fusarium culmorum, which is a pathogenic fungi that cause decomposition of roots of vegetables. Accordingly, chitinase enzyme activity assay was performed on 31 isolates that have an antifungal activity, and among them the isolate of Bacillus subtilis TV-125 was selected, which has demonstrated the highest activity. Chitinase enzyme was purified by using ammonium sulphate and DEAE-sephadex ion exchange chromatography. Ammonium sulphate precipitation of chitinase enzyme from Bacillus subtilis TV-125 isolate was performed at maximum range of 0-20%, and 28.4-fold purification was obtained with a 13.4% of yield. Optimum activity of the purified enzyme was observed at pH 4.0 and at 50°C of temperature. In addition, it was identified that Bacillus subtilis TV-125A isolate retains 42% of its activity at 80°C temperature. In the last phase of the study, chitinase enzyme purified from Bacillus subtilis TV-125A was tested on four fungal agents, although all the results were positive, it was particularly effective on F. culmorum according to the findings.

Journal ArticleDOI
TL;DR: WAMPs represent novel protease inhibitors that are active against fungal metalloproteases, suggesting that fungalysin plays an important role in fungal development and a novel molecular mechanism of dynamic interplay between host defense molecules and fungal virulence factors is suggested.
Abstract: The multilayered plant immune system relies on rapid recognition of pathogen-associated molecular patterns followed by activation of defense-related genes, resulting in the reinforcement of plant cell walls and the production of antimicrobial compounds To suppress plant defense, fungi secrete effectors, including a recently discovered Zn-metalloproteinase from Fusarium verticillioides, named fungalysin Fv-cmp This proteinase cleaves class IV chitinases, which are plant defense proteins that bind and degrade chitin of fungal cell walls In this study, we investigated plant responses to such pathogen invasion, and discovered novel inhibitors of fungalysin We produced several recombinant hevein-like antimicrobial peptides named wheat antimicrobial peptides (WAMPs) containing different amino acids (Ala, Lys, Glu, and Asn) at the nonconserved position 34 An additional Ser at the site of fungalysin proteolysis makes the peptides resistant to the protease Moreover, an equal molar concentration of WAMP-1b or WAMP-2 to chitinase was sufficient to block the fungalysin activity, keeping the chitinase intact Thus, WAMPs represent novel protease inhibitors that are active against fungal metalloproteases According to in vitro antifungal assays WAMPs directly inhibited hyphal elongation, suggesting that fungalysin plays an important role in fungal development A novel molecular mechanism of dynamic interplay between host defense molecules and fungal virulence factors is suggested

Journal ArticleDOI
TL;DR: A chitinase and a chitosanase were induced from a squid pen powder (SPP)-containing medium of Bacillus cereus TKU030 and purified by precipitation with ammonium sulphate and combined column chromatography and showed high stability in the presence of various surfactants, such as SDS,Tween 20, Tween 40 and Triton X-100.

Journal ArticleDOI
TL;DR: The chitinolytic strain B. thuringiensis UM96 was able to protect Medicago truncatula plants in vitro from B. cinerea infection and significantly reduced the necrotic zones and root browning of the plants.
Abstract: The potential use of Bacillus thuringiensis UM96 as a biocontrol agent for the grey mould phytopathogen Botrytis cinerea was evaluated. In order to dissect the mode of action of this UM96 strain, we also examined the role of lytic activities in the antagonism. First, B. thuringiensis UM96 was characterised based on 16S rRNA and gyrA gene sequencing and phenotypic traits. Petri dish biocontrol assays demonstrated that when strain UM96 was inoculated 24 h previous to B. cinerea, the mycelial growth was inhibited by up to 70%. Test for lytic enzymes activities of cellulase and glucanase was negative. Chitinase was the only positive enzyme activity in two different culture media. PCR detection of the chiB gene was also positive. Chitinolytic supernatants, obtained from rich and minimal media supplemented with colloidal chitin as the sole carbon source, from B. thuringiensis UM96 showed a strong inhibitory effect of B. cinerea that was not observed with heat-treated supernatant. Interestingly, when the superna...

Journal ArticleDOI
22 Sep 2014-PLOS ONE
TL;DR: Evidence is provided to show that ChiA2 is important for V. cholerae survival in intestine as well as in pathogenesis, and it is demonstrated that chiA2 de-glycosylates mucin and releases reducing sugars like GlcNAc and its oligomers, which are helpful for growth and survival in the host intestine.
Abstract: In aquatic environments, Vibrio cholerae colonizes mainly on the chitinous surface of copepods and utilizes chitin as the sole carbon and nitrogen source. Of the two extracellular chitinases essential for chitin utilization, the expression of chiA2 is maximally up-regulated in host intestine. Recent studies indicate that several bacterial chitinases may be involved in host pathogenesis. However, the role of V. cholerae chitinases in host infection is not yet known. In this study, we provide evidence to show that ChiA2 is important for V. cholerae survival in intestine as well as in pathogenesis. We demonstrate that ChiA2 de-glycosylates mucin and releases reducing sugars like GlcNAc and its oligomers. Deglycosylation of mucin corroborated with reduced uptake of alcian blue stain by ChiA2 treated mucin. Next, we show that V. cholerae could utilize mucin as a nutrient source. In comparison to the wild type strain, ΔchiA2 mutant was 60-fold less efficient in growth in mucin supplemented minimal media and was also ∼6-fold less competent to survive when grown in the presence of mucin-secreting human intestinal HT29 epithelial cells. Similar results were also obtained when the strains were infected in mice intestine. Infection with the ΔchiA2 mutant caused ∼50-fold less fluid accumulation in infant mice as well as in rabbit ileal loop compared to the wild type strain. To see if the difference in survival of the ΔchiA2 mutant and wild type V. cholerae was due to reduced adhesion of the mutant, we monitored binding of the strains on HT29 cells. The initial binding of the wild type and mutant strain was similar. Collectively these data suggest that ChiA2 secreted by V. cholerae in the intestine hydrolyzed intestinal mucin to release GlcNAc, and the released sugar is successfully utilized by V. cholerae for growth and survival in the host intestine.

Journal ArticleDOI
11 Jun 2014-PLOS ONE
TL;DR: A distinct type of CTLs that may have novel functions specific to the development of the gelatinous (G-type) cellulosic walls is defined.
Abstract: Plant chitinases (EC 3.2.1.14) and chitinase-like (CTL) proteins have diverse functions including cell wall biosynthesis and disease resistance. We analyzed the expression of 34 chitinase and chitinase-like genes of flax (collectively referred to as LusCTLs), belonging to glycoside hydrolase family 19 (GH19). Analysis of the transcript expression patterns of LusCTLs in the stem and other tissues identified three transcripts (LusCTL19, LusCTL20, LusCTL21) that were highly enriched in developing bast fibers, which form cellulose-rich gelatinous-type cell walls. The same three genes had low relative expression in tissues with primary cell walls and in xylem, which forms a xylan type of secondary cell wall. Phylogenetic analysis of the LusCTLs identified a flax-specific sub-group that was not represented in any of other genomes queried. To provide further context for the gene expression analysis, we also conducted phylogenetic and expression analysis of the cellulose synthase (CESA) family genes of flax, and found that expression of secondary wall-type LusCESAs (LusCESA4, LusCESA7 and LusCESA8) was correlated with the expression of two LusCTLs (LusCTL1, LusCTL2) that were the most highly enriched in xylem. The expression of LusCTL19, LusCTL20, and LusCTL21 was not correlated with that of any CESA subgroup. These results defined a distinct type of CTLs that may have novel functions specific to the development of the gelatinous (G-type) cellulosic walls.

Journal ArticleDOI
TL;DR: It is found that heat shock at 42 °C in Saccharomyces cerevisiae and treatment of this yeast species and Candida albicans with the antifungal drug caspofungin resulted in 2- to 3-fold increase of chitin and in a reduction of β-(1,3)-glucan accompanied by an increase of β- glucan, whereas ethanol stress had apparently no effect on yeast cell wall composition.
Abstract: A reliable method to determine cell wall polysaccharides composition in yeast is presented, which combines acid and enzymatic hydrolysis. Sulphuric acid treatment is used to determine mannans, whereas specific hydrolytic enzymes are employed in a two sequential steps to quantify chitin and the proportion of β-(1, 3) and β-(1, 6)-glucan in the total β-glucan of the cell wall. In the first step, chitin and β-(1, 3)-glucan were hydrolysed into their corresponding monomers N -acetylglucosamine and glucose, respectively, by the combined action of a chitinase from Streptomyces griseus and a pure preparation of endo/exo-β-(1, 3)-glucanase from Trichoderma species . This step was followed by addition of recombinant endo-β-(1, 6)-glucanase from Trichoderma harzianum with β-glucosidase from Aspergillus niger to hydrolyse the remaining β-glucan. This latter component corresponded to a highly branched β-(1, 6)-glucan that contained about 75–80% of linear β-(1, 6)-glucose linked units as deduced from periodate oxidation. We validated this novel method by showing that the content of β-(1, 3), β-(1, 6)-glucan or chitin was dramatically decreased in yeast mutants defective in the biosynthesis of these cell wall components. Moreover, we found that heat shock at 42 °C in Saccharomyces cerevisiae and treatment of this yeast species and Candida albicans with the antifungal drug caspofungin resulted in 2- to 3-fold increase of chitin and in a reduction of β-(1, 3)-glucan accompanied by an increase of β-(1, 6)-glucan, whereas ethanol stress had apparently no effect on yeast cell wall composition.

Journal ArticleDOI
TL;DR: Mechanisms of action and effectiveness of the antifungal yeast Cryptococcus laurentii, strain L5D, were examined against the causal agent of anthracnose Colletotrichum gloeosporioides in mango, showing high antagonistic potential in vivo and competition for nutrients, specifically for sucrose.

Journal ArticleDOI
TL;DR: It is reported that a series of fully deacetylated chitooligosaccharides (GlcN)2–7 can act as inhibitors against the insect chitinase OfChtI, the human chitInase HsCht, and the bacterial chit inases SmChiA and SmchiB with IC50 values at micromolar to millimolar levels.

Journal ArticleDOI
TL;DR: A chit inase from Aeromonas veronii B565, designated ChiB565, is described, which potently degrades shrimp-shell chitin and resists proteolysis and is optimally active at pH 5.0 and 50 °C and stable between pH 4.5 and 9.0.
Abstract: Chitin, present in crustacean shells, insects, and fungi, is the second most plentiful natural organic fiber after wood. To effectively use chitin in a cost-saving and environmentally friendly way in aquaculture, crustacean shells (e.g., shrimp-shell meal) are supplemented into aquafeed after degradation by chemical methods. Herein, we describe a chitinase from Aeromonas veronii B565, designated ChiB565, which potently degrades shrimp-shell chitin and resists proteolysis. We isolated recombinant ChiB565 of the expected molecular mass in large yield from Pichia pastoris. ChiB565 is optimally active at pH 5.0 and 50 °C and stable between pH 4.5 and 9.0 at 50 °C and below. Compared with the commercial chitinase C-6137, which cannot degrade shrimp-shell chitin, ChiB565 hydrolyzes shrimp-shell chitin in addition to colloidal chitin, powdered chitin, and β-1,3-1,4-glucan. The optimal enzyme concentration and reaction time for in vitro degradation of 0.1 g of powdered shrimp shell are 30 U of ChiB565 and 3 h, respectively. A synergistic protein-release effect occurred when ChiB565 and trypsin were incubated in vitro with shrimp shells. Tilapia were fed an experimental diet containing 5 % (w/w) shrimp bran and 16.2 U/kg ChiB565, which significantly improved growth and feed conversion compared with a control diet lacking ChiB565. Dietary ChiB565 enhanced nitrogen digestibility and downregulated intestinal IL-1β expression. The immunologically relevant protective effects of dietary ChiB565 were also observed for 2 to 3 days following exposure to pathogenic Aeromonas hydrophila.

Journal ArticleDOI
TL;DR: The enzyme exhibited significant activity up to 3% salt concentration, indicating saline nature; its activity was enhanced with calcium, potassium and magnesium; whereas copper and mercury were found to be inhibitory.
Abstract: An alkaline chitinase was purified from the bacterium Paenibacillus pasadenensis NCIM 5434 isolated from alkaline littoral soil of Lonar Lake. The chitinase was purified by ammonium sulfate precipitation followed by DEAE cellulose column chromatography. Enzyme was purified by 8.87 folds with 24.96% yield. Molecular characterization through SDS–PAGE analysis showed that it has molecular weight of about ∼35 kDa. The enzyme kinetics studies of purified chitinase revealed the following characteristics, Km 6.25 mg ml−1 and Vmax 434.78 µM for colloidal chitin as a substrate. The chitinase showed optimum pH 10 and temperature 37 °C. The enzyme exhibited significant activity up to 3% salt concentration, indicating saline nature. Its activity was enhanced with calcium, potassium and magnesium; whereas copper and mercury were found to be inhibitory. Since, it showed antifungal activity against Penicillium and Aspergillus, it could be used as powerful biocontrol agent.

Journal ArticleDOI
TL;DR: The first crystal structure of an insect chitinase that is indispensable to moulting is revealed and it is shown that it has a Tournaisian structure similar to that of the H2O2 molecule.
Abstract: Insects possess a greater number of chitinases than any other organisms. This work is the first report of unliganded and oligosaccharide-complexed crystal structures of the insect chitinase OfChtI from Ostrinia furnacalis, which is essential to moulting. The obtained crystal structures were solved at resolutions between 1.7 and 2.2 A. A structural comparison with other chitinases revealed that OfChtI contains a long substrate-binding cleft similar to the bacterial chitinase SmChiB from Serratia marcescens. However, unlike the exo-acting SmChiB, which has a blocked and tunnel-like cleft, OfChtI possesses an open and groove-like cleft. The complexed structure of the catalytic domain of OfChtI (OfChtI-CAD) with (GlcNAc)2/3 indicates that the reducing sugar at subsite −1 is in an energetically unfavoured `boat' conformation, a state that possibly exists just before the completion of catalysis. Because OfChtI is known to act from nonreducing ends, (GlcNAc)3 would be a hydrolysis product of (GlcNAc)6, suggesting that OfChtI possesses an endo enzymatic activity. Furthermore, a hydrophobic plane composed of four surface-exposed aromatic residues is adjacent to the entrance to the substrate-binding cleft. Mutations of these residues greatly impair the chitin-binding activity, indicating that this hydrophobic plane endows OfChtI-CAD with the ability to anchor chitin. This work reveals the unique structural characteristics of an insect chitinase.

Journal Article
TL;DR: The present review focuses on recent advances in fungal chitinases, containing a short introduction to types of chit inases, their fermentative production, purification and characterization and molecular cloning and expression.
Abstract: After cellulose, chitin is the second most abundant organic and renewable polysaccharide in nature. This polymer is degraded by enzymes called chitinases which are a part of the glycoside hydrolase family. Chitinases have many important biophysiological functions and immense potential applications especially in control of phytopathogens, production of chito- oligosaccharides with numerous uses and in treatment and degradation of chitinous biowaste. At present many microbial sources are being explored and tapped for chitinase production which includes potential fungal cultures. With advancement in molecular biology and gene cloning techniques, research on fungal chitinases have made fast progress. The present review focuses on recent advances in fungal chitinases, containing a short introduction to types of chitinases, their fermentative production, purification and characterization and molecular cloning and expression.

Journal ArticleDOI
TL;DR: A holin and an endopeptidase operate in tandem as components of a protein secretion system used by the gram-negative bacterium Serratia marcescens to secrete the chitinolytic machinery.
Abstract: Pathogenic bacteria adapt to their environment and manipulate the biochemistry of hosts by secretion of effector molecules. Serratia marcescens is an opportunistic pathogen associated with healthcare-acquired infections and is a prolific secretor of proteins, including three chitinases (ChiA, ChiB, and ChiC) and a chitin binding protein (Cbp21). In this work, genetic, biochemical, and proteomic approaches identified genes that were required for secretion of all three chitinases and Cbp21. A genetic screen identified a holin-like protein (ChiW) and a putative l-alanyl-d-glutamate endopeptidase (ChiX), and subsequent biochemical analyses established that both were required for nonlytic secretion of the entire chitinolytic machinery, with chitinase secretion being blocked at a late stage in the mutants. In addition, live-cell imaging experiments demonstrated bimodal and coordinated expression of chiX and chiA and revealed that cells expressing chiA remained viable. It is proposed that ChiW and ChiX operate in tandem as components of a protein secretion system used by gram-negative bacteria.

Journal ArticleDOI
TL;DR: Solid state fermentation is a bioprocess that would produce at great scale enzymes and some other metabolites in grade of increasing the entomopathogenic fungi virulence, in the control of insects and potentially in some diseases affecting plants.
Abstract: Entomopathogenic fungi (EF) are recognized biological control agents of insects. Basically, the entomopathogenic fungi pathogen activity depends on the ability of its enzymatic equipment, consisting of lipases, proteases and chitinases, which are in charge of breaking down the insect’s integument. Lipases are the first enzymes synthesized by the entomopathogenic fungi. Recently, a cytochrome P450 subfamily, referred as CYP52XI and MrCYP52 has been identified in Beauveria bassiana and Metarhizium robertsii, respectively. These break down long-chain alkenes and fatty acids to become initial nutrients. Subsequently, subtilisin type (Pr1) proteases sintetize; these enzymes are considered as virulence indicators and they are regulated by a signal transduction mechanism activated by the protein kinase A (PKA) mediated by AMPc. Through the employment of genetic engineering, it has been possible to increase virulence producing Pr1 recombinants with Androctonus australis neurotoxins or with chitinases, reducing the insect’s time of death. In the course of time, the Pr1 protease gene has presented evolutionary adaptations by gene duplication or horizontal transfer infecting different orders of insects. In the same way, the entomopathogenic fungi chitinases have presented a functional diversification. Currently, these have been phylogenetically classified into three subgroups, in accordance to the catalytic site domain and the chitin binding domain. The chitinolytic activity has increased through a directed evolution processes and genetic recombination with Bombyx mori chitinase. Recently, enzymes have been employed as control agents for insects and phytopathogenic fungi (disease originator) opening new potentialities in order to improve the entomopathogenic fungi use. Solid state fermentation is a bioprocess that would produce at great scale enzymes and some other metabolites in grade of increasing the entomopathogenic fungi virulence, in the control of insects and potentially in some diseases affecting plants.

Journal ArticleDOI
TL;DR: Six chitinase genes in the oriental river prawn, Macrobrachium nipponense are identified according to the established expressed sequence tag (EST) information using Rapid Amplification of the cDNA Ends (RACE) technique and homology cloning and their expression in different tissues and different developmental stages suggested that all of them have a function in the digestion of chitinous foods, modification of gut peritrophic membrane and degradation of the Chitin exoskeleton.
Abstract: Chitinase plays crucial physiological roles in crustaceans, including the digestion of chitin-containing food, moulting and the defense of shrimp against viruses. However, in contrast to insect species, no genome-wide analysis has been carried out in crustacean species and cDNAs encoding chitinase and chitinase-like proteins have been characterized in relatively few species. In this study, we identified six chitinase genes in the oriental river prawn, Macrobrachium nipponense, according to the established expressed sequence tag (EST) information using Rapid Amplification of the cDNA Ends (RACE) technique and homology cloning. We assigned these genes to three different chitinase groupings, which were designated MnCht1A, 1B, 3A, 3B, 3C and 4. The domain organization analysis of the six MnCht proteins revealed that only MnCht3C and MnCht4 possessed full structure, while MnCht1A, 1B, 3A and 3B lacked the serine/threonine (S/T)-rich linker and chitin-binding domains (CBDs). Their expression in different tissues and different developmental stages suggested that all of them have a function in the digestion of chitinous foods, modification of gut peritrophic membrane and degradation of the chitin exoskeleton. Analysis of the stage-specific moulting cycle and different temperature stimulation provided further evidence that MnCht1A, 1B and 3B have pivotal roles in the moulting cycle, while MnCht 4 only assists in the moulting process. This study provides important information for further investigations on the functions of chitinase in M. nipponense and other crustaceans.

Journal ArticleDOI
TL;DR: The HschiA1 gene of the archaeon Halobacterium salinarum CECT 395 was cloned and overexpressed as an active protein of 66.5 kDa in Escherichia coli and can be identified as an exo-acting enzyme with potential interest regarding the biodegradation of chitin waste or its bioconversion into biologically active products.
Abstract: The HschiA1 gene of the archaeon Halobacterium salinarum CECT 395 was cloned and overexpressed as an active protein of 66.5 kDa in Escherichia coli. The protein called HsChiA1p has a modular structure consisting of a glycosyl hydrolase family 18 catalytic region, as well as a N-terminal family 5 carbohydrate-binding module and a polycystic kidney domain. The purified recombinant chitinase displayed optimum catalytic activity at pH 7.3 and 40 °C and showed high stability over broad pH (6-8.5) and temperature (25-45 °C) ranges. Protein activity was stimulated by the metal ions Mg(+2), K(+), and Ca(+2) and strongly inhibited by Mn(+2). HsChiA1p is salt-dependent with its highest activity in the presence of 1.5 M of NaCl, but retains 20% of its activity in the absence of salt. The recombinant enzyme hydrolysed p-NP-(GlcNAc)3, p-NP-(GlcNAc), crystalline chitin, and colloidal chitin. From its sequence features and biochemical properties, it can be identified as an exo-acting enzyme with potential interest regarding the biodegradation of chitin waste or its bioconversion into biologically active products.

Reference EntryDOI
15 Aug 2014
TL;DR: Biotechnological applications of chitin derivatives, such as chitosan and chito-oligosaccharides, are currently an expanding area in biomedicine, pharmaceutical and food technology and agro-biosciences.
Abstract: Chitin is a naturally occurring fibre-forming polymer that plays a protective role in many lower eukaryotes similar to that of cellulose in plants. Chemically it is a long-chain unbranched polysaccharide made of N-acetylglucosamine residues linked through β-1,4 covalent bonds; it is the second most abundant organic compound in nature, after cellulose. Taking into account the role played by chitin in different biological structures (i.e. fungal cell walls, insect peritrophic matrix, insect and crustacean cuticles, eggshells from nematodes, cyst wall of protozoa), its metabolism (biosynthesis and degradation) is essential for different morphogenetic events. Absent in vertebrates and plants, chitin participates in host–parasite interactions and represents a parasite-specific target for chemotherapeutic attack and also plays a role in host immune responses. Because of its abundance in nature and its properties, biotechnological applications of chitin derivatives, such as chitosan and chito-oligosaccharides, are currently an expanding area in biomedicine, pharmaceutical and food technology and agro-biosciences. Key Concepts: Chitin is a long-chain unbranched polysaccharide made of β-1,4-linked anhydro-2-acetamido-2-deoxy-d-glucose (GlcNAc) which forms crystalline fibrillar structures following association of adjacent chains through hydrogen bonds between the N–H and the CO groups. Nascent chitin is a growing chitin chain which is being synthesised by the chitin synthase and it represents a good substrate for chitinolytic enzymes. Microfibrillar chitin is a crystalline structure formed by chains of the polysaccharide which associate through hydrogen bonds between adjacent chains and it is responsible for the physico-chemical properties of the polymer. The fungal cell wall is a supramolecular network outside the plasma membrane, formed by structural polysaccharides, including chitin, and proteins and glycoproteins, that protects the fungal cell and determines morphology, similarly to cuticle (exosqueleton) in insects. Insect cuticle (exosqueleton) is an extracellular matrix covering the epidermis and trachea, composed mainly by chitin (and proteins), which protects the animal and confers morphology. The peritrophic membrane (matrix) is an extracellular layer that covers the midgut in most arthropods and it is made of chitin, proteins and proteoglycans, and provides protection to the underlaying digestive cells. Chitin biosynthesis is a strongly regulated process, both spatially and temporally, as chitin deposition is essential for fungal growth and development (moulting) in arthropods. Chitin synthases are membrane bound enzymes that incorporate the substrate (UDP-N-acetylglucosamine) from the cytosol to the nascent chitin chain that is extruded outside the membrane. Chitinases are chitin-hydrolysing enzymes that play important roles in the physiology of chitin-containing eukaryotes, and chitinolytic bacteria are active in a scavenging role by degrading massive amounts of chitin in marine and soil biomass, avoiding its accumulation and favouring the utilisation of chitin as a renewable source. The absence of chitin in vertebrates and plants makes the chitin metabolism a potentially useful parasite-specific target for chemotherapeutic attack. In mammals, chitin regulates immune responses playing a role in inflammation, and allergic diseases. In plants, chitin elicits defence responses, and in leguminous plants, chitin oligosaccharides produced by rhizobia promote plant nodulation. Owing to their abundance in nature and properties, chitin, chitosan and their derivatives have gained potential interest for a wide range of areas, including biopharmaceutical and biomedical applications. Keywords: polysaccharide; chitosan; chitinase; chitin synthetase

Journal ArticleDOI
TL;DR: This study, was the first showing the antimicrobial efficiency of moderately halophilic bacteria by means of their intracellular compounds, by Means of the spore germination reduction and the destroy of mycelial growth of Botrytis cinerea, in vitro.
Abstract: In the present study, we firstly aimed to determine the ability of halophilic bacteria to improve tomato growth. as well as to detect the antimicrobial activities from two moderately halophilic bacteria strain M3-23 of Virgibacillus marismortui and strain J31 of Terribacillus halophilus exhibited by their intracellular proteins. The results showed that both bacteria were able to improve stem tomato growth by comparison of untreated tomato. The halophilic bacteria were also able to produce intracellular antifungal enzyme: glucanase produced by V. marismortui (1.74U/mg) and chitinase (39.39U/mg) produced by T. halophilus. Both chitinases were halotolerants (active in the presence of (0% to 30% NaCl (w/v)). Chitinase produced by strain J31 was alcaline (pH optimum pH 12), but chitinase from strain M3- 23 was acidic (pH 4 optimum) more than 90% and 80% of activities were retained in the presence of pH value from 4 to 12, respectively for strain J31 and M3-23. Both enzymes were thermotolerants; optimum temperature was 80°C and 90°C respectively for strain J31 and strain M3-23. Both strains have lysozyme activity and value ranging from 6.6 U/ml to 6.8 U/ml respectively for strain J31 and strain M3-23. On the whole, the most potent in vitro antifungal effect was demonstrated by intracellular compound produced by strain J31 compared to strain M3-23. This study, was the first showing the antimicrobial efficiency of moderately halophilic bacteria by means of their intracellular compounds, by means of the spore germination reduction and the destroy of mycelial growth of Botrytis cinerea, in vitro. The distinguishable characteristics of their intracellular halotolerant and thermotolerant chitinases make them as good candidates for biotechnological applications.

Journal ArticleDOI
24 Mar 2014-PLOS ONE
TL;DR: The results support a key role of the chitinase(s) in biofilm formation through modulation of the bacterial surface properties and position chit inase as a potential anti-biofilm enzyme in Francisella species.
Abstract: Biofilms, multicellular communities of bacteria, may be an environmental survival and transmission mechanism of Francisella tularensis. Chitinases of F. tularensis ssp. novicida (Fn) have been suggested to regulate biofilm formation on chitin surfaces. However, the underlying mechanisms of how chitinases may regulate biofilm formation are not fully determined. We hypothesized that Fn chitinase modulates bacterial surface properties resulting in the alteration of biofilm formation. We analyzed biofilm formation under diverse conditions using chitinase mutants and their counterpart parental strain. Substratum surface charges affected biofilm formation and initial attachments. Biophysical analysis of bacterial surfaces confirmed that the chi mutants had a net negative-charge. Lectin binding assays suggest that chitinase cleavage of its substrates could have exposed the concanavalin A-binding epitope. Fn biofilm was sensitive to chitinase, proteinase and DNase, suggesting that Fn biofilm contains exopolysaccharides, proteins and extracellular DNA. Exogenous chitinase increased the drug susceptibility of Fn biofilms to gentamicin while decreasing the amount of biofilm. In addition, chitinase modulated bacterial adhesion and invasion of A549 and J774A.1 cells as well as intracellular bacterial replication. Our results support a key role of the chitinase(s) in biofilm formation through modulation of the bacterial surface properties. Our findings position chitinase as a potential anti-biofilm enzyme in Francisella species.