Showing papers on "Chitinase published in 2013"
TL;DR: It is found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability and radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of thecell wall.
Abstract: Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae.
363 citations
TL;DR: The catalytic mechanisms of these enzymes as well as the structural basis of each enzyme's specific role in the chitin degradation process are discussed, and how knowledge of this enzyme system may be extrapolated to other enzyme systems for conversion of insoluble polysaccharides is discussed.
Abstract: The chitinolytic machinery of Serratia marcescens is one of the best known enzyme systems for the conversion of insoluble polysaccharides This machinery includes four chitin-active enzymes: ChiC, an endo-acting non-processive chitinase; ChiA and ChiB, two processive chitinases moving along chitin chains in opposite directions; and CBP21, a surface-active CBM33-type lytic polysaccharide monooxygenase that introduces chain breaks by oxidative cleavage Furthermore, an N-acetylhexosaminidase or chitobiase converts the oligomeric products from the other enzymes to monomeric N-acetylglucosamine Here we discuss the catalytic mechanisms of these enzymes as well as the structural basis of each enzyme's specific role in the chitin degradation process We also discuss how knowledge of this enzyme system may be extrapolated to other enzyme systems for conversion of insoluble polysaccharides, in particular conversion of cellulose by cellulases and GH61-type lytic polysaccharide monooxygenases
243 citations
TL;DR: Chitinase enzyme has received increased attention due to its wide range of biotechnological applications, especially in agriculture for biocontrol of phytopathogenic fungi and harmful insects, and out of metal ions Mn 2+ and Cu 2+ enhanced enzyme production in the case of HS6, however, Co 2+ was the most appropriate for HS4.
182 citations
TL;DR: Different aspects of chitinases and chi-lectins from bacteria, fungi, insects, plants and mammals are discussed.
172 citations
TL;DR: The data revealed that chitin amendment had raised the suppressiveness of soil, in particular toward Verticillium dahliae, 9 months after the (second) treatment, extending to 2 years following treatment, and major effects of the added Chitin on the soil microbial communities were detected.
Abstract: A long-term experiment on the effect of chitin addition to soil on the suppression of soilborne pathogens was set up and monitored for 8 years in an experimental field, Vredepeel, The Netherlands. Chitinous matter obtained from shrimps was added to soil top layers on two different occasions, and the suppressiveness of soil toward Verticillium dahliae, as well as plant-pathogenic nematodes, was assessed, in addition to analyses of the abundances and community structures of members of the soil microbiota. The data revealed that chitin amendment had raised the suppressiveness of soil, in particular toward Verticillium dahliae, 9 months after the (second) treatment, extending to 2 years following treatment. Moreover, major effects of the added chitin on the soil microbial communities were detected. First, shifts in both the abundances and structures of the chitin-treated soil microbial communities, both of total soil bacteria and fungi, were found. In addition, the abundances and structures of soil actinobacteria and the Oxalobacteraceae were affected by chitin. At the functional gene level, the abundance of specific (family-18 glycoside hydrolase) chitinase genes carried by the soil bacteria also revealed upshifts as a result of the added chitin. The effects of chitin noted for the Oxalobacteraceae were specifically related to significant upshifts in the abundances of the species Duganella violaceinigra and Massilia plicata. These effects of chitin persisted over the time of the experiment.
130 citations
TL;DR: Three parameters were evaluated after interaction between four Trichoderma species and plant-pathogenic fungi: Fusarium solani, Rhizoctonia solani and Sclerotinia sclerotiorum.
Abstract: Trichoderma spp. are used for biocontrol of several plant pathogens. However, their efficient interaction with the host needs to be accompanied by production of secondary metabolites and cell wall-degrading enzymes. Three parameters were evaluated after interaction between four Trichoderma species and plant-pathogenic fungi: Fusarium solani, Rhizoctonia solani and Sclerotinia sclerotiorum. Trichoderma harzianum and T. asperellum were the most effective antagonists against the pathogens. Most of the Trichoderma species produced toxic volatile metabolites, having significant effects on growth and development of the plant pathogens. When these species were grown in liquid cultures with cell walls from these plant pathogens, they produced and secreted β-1,3-glucanase, NAGAse, chitinase, acid phosphatase, acid proteases and alginate lyase.
117 citations
TL;DR: It is proposed that surface stresses lead to a conserved cell wall remodeling response that is mainly governed by Mkc1 and is characterized by chitin reinforcement of the wall and the expression of remedial wall remodelling enzymes.
Abstract: The human fungal pathogen Candida albicans can grow at temperatures of up to 45°C. Here, we show that at 42°C substantially less biomass was formed than at 37°C. The cells also became more sensitive to wall-perturbing compounds, and the wall chitin levels increased, changes that are indicative of wall stress. Quantitative mass spectrometry of the wall proteome using 15N metabolically labeled wall proteins as internal standards revealed that at 42°C the levels of the β-glucan transglycosylases Phr1 and Phr2, the predicted chitin transglycosylases Crh11 and Utr2, and the wall maintenance protein Ecm33 increased. Consistent with our previous results for fluconazole stress, this suggests that a wall-remodeling response is mounted to relieve wall stress. Thermal stress as well as different wall and membrane stressors led to an increased phosphorylation of the mitogen-activated protein (MAP) kinase Mkc1, suggesting activation of the cell wall integrity (CWI) pathway. Furthermore, all wall and membrane stresses tested resulted in diminished cell separation. This was accompanied by decreased secretion of the major chitinase Cht3 and the endoglucanase Eng1 into the medium. Consistent with this, cht3 cells showed a similar phenotype. When treated with exogenous chitinase, cell clusters both from stressed cells and mutant strains were dispersed, underlining the importance of Cht3 for cell separation. We propose that surface stresses lead to a conserved cell wall remodeling response that is mainly governed by Mkc1 and is characterized by chitin reinforcement of the wall and the expression of remedial wall remodeling enzymes.
98 citations
TL;DR: The potential of rice chitinase genes to enhance resistance against M. fijiensis in banana was demonstrated as well as the usefulness of the leaf disk bioassay for early disease screening in transgenic banana lines.
Abstract: Transgenic banana (Musa acuminata ‘Gros Michel’) integrating either of two rice chitinase genes was generated and its resistance to Black Leaf Streak disease caused by the fungus Mycosphaerella fijiensis was tested using a leaf disk bioassay. PCR screening indicated the presence of the hpt selectable marker gene in more than 90 % of the lines tested, whereas more than three quarters of the lines contained the linked rice chitinase gene resulting in a co-transformation frequency of at least 71.4 %. Further, a unique stable integration of the transgenes in each line revealed some false negative PCR results and the expected co-transformation frequency of 100 %. The transgene insert number per line ranged from 1 to 5 and single transgene insert lines (25 % of all) were identified. Considerable delay in disease development (up to 63 days post-incoculation) over a monitoring period of 108 days occurred in nine lines with extracellularly targeted chitinase out of 17 transgenic lines tested and their necrotic leaf area decreased by 73–94 % compared to the untransformed susceptible control line. Finally, correlation between symptom development and rice chitinase expression was confirmed in two lines by Western analysis. The potential of rice chitinase genes to enhance resistance against M. fijiensis in banana was demonstrated as well as the usefulness of the leaf disk bioassay for early disease screening in transgenic banana lines.
98 citations
TL;DR: The results of this study enhance understanding of the response of the soil bacterial communities to chitin and are of use for both the understanding of soil suppressiveness and the possible mining of soil for novel enzymes.
Abstract: Chitin amendment is a promising soil management strategy that may enhance the suppressiveness of soil toward plant pathogens. However, we understand very little of the effects of added chitin, including the putative successions that take place in the degradative process. We performed an experiment in moderately acid soil in which the level of chitin, next to the pH, was altered. Examination of chitinase activities revealed fast responses to the added crude chitin, with peaks of enzymatic activity occurring on day 7. PCR-denaturing gradient gel electrophoresis (DGGE)-based analyses of 16S rRNA and chiA genes showed structural changes of the phylogenetically and functionally based bacterial communities following chitin addition and pH alteration. Pyrosequencing analysis indicated (i) that the diversity of chiA gene types in soil is enormous and (i) that different chiA gene types are selected by the addition of chitin at different prevailing soil pH values. Interestingly, a major role of Gram-negative bacteria versus a minor one of Actinobacteria in the immediate response to the added chitin (based on 16S rRNA gene abundance and chiA gene types) was indicated. The results of this study enhance our understanding of the response of the soil bacterial communities to chitin and are of use for both the understanding of soil suppressiveness and the possible mining of soil for novel enzymes.
93 citations
TL;DR: Crops such as tomato, potato, maize, groundnut, mustard, finger millet, cotton, lychee, banana, grape, wheat and rice have been successfully engineered for fungal resistance either with chitinase alone or in combination with other PR proteins.
Abstract: Crop plants have evolved an array of mechanisms to counter biotic and abiotic stresses. Many pathogenesis-related proteins are expressed by plants during the attack of pathogens. Advances in recombinant DNA technology and understanding of plant-microbe interactions at the molecular level have paved the way for isolation and characterization of genes encoding such proteins, including chitinases. Chitinases are included in families 18 and 19 of glycosyl hydrolases (according to www.cazy.org ) and they are further categorized into seven major classes based on their aminoacid sequence homology, three-dimensional structures, and hydrolytic mechanisms of catalytic reactions. Although chitin is not a component of plant cell walls, plant chitinases are involved in development and non-specific stress responses. Also, chitinase genes sourced from plants have been successfully over-expressed in crop plants to combat fungal pathogens. Crops such as tomato, potato, maize, groundnut, mustard, finger millet, cotton, lychee, banana, grape, wheat and rice have been successfully engineered for fungal resistance either with chitinase alone or in combination with other PR proteins.
90 citations
TL;DR: The biochemical approach described in this article with E. sativa provide the basis for further efforts concentrating on the isolation and characterization of elements involved in perception and in the early steps of intracellular signal transduction.
Abstract: Plants have developed many mechanisms to protect themselves against most potential microbial pathogens and diseases. Pathogenesis-related proteins are produced as a part of the active defenses to prevent attack. In this study, the induction of PR proteins in Eruca sativa in response to fungal pathogen Alternaria brassicicola was investigated in 10 days and one-month-old plants. Induction of pathogen resulted in a much marked increase in the activities of β-1,3-glucanase and chitinase in resistant cultivar (RTM-2002) as compared to susceptible (T-27) one. The enzyme activity gradually increased throughout the experimental period of 168 h compare to control. However, the activation of β-1,3-glucanase and chitinase was more rapid and to a greater extent in plants of RTM-2002 than in T-27. western blot analysis revealed the presence of 33 and 32 kDa β-1,3-glucanase and chitinase in induced arugula plants, respectively. The biochemical approach described in this article with E. sativa provide the basis for fur...
TL;DR: It is revealed that sustained and timely induction and accumulation of these defense enzymes and PR-proteins enhance the resistance in groundnut against leafminer insect and collar rot disease.
TL;DR: A chitinase gene from rice (Rchit) was introduced into three varieties of peanut through Agrobacterium-mediated genetic transformation resulting in 30 transgenic events harboring the Rchit gene, revealing a Mendelian inheritance pattern for the transgenes.
Abstract: A chitinase gene from rice (Rchit) was introduced into three varieties of peanut through Agrobacterium-mediated genetic transformation resulting in 30 transgenic events harboring the Rchit gene. Stable integration and expression of the transgenes were confirmed using PCR, RT-PCR and Southern blot analysis. Progeny derived from selfing of the primary transgenic events revealed a Mendelian inheritance pattern (3:1) for the transgenes. The chitinase activity in the leaves of the transgenic events was 2 to 14-fold greater than that in the non-transformed control plants. Seeds of most transgenic events showed 0–10 % A. flavus infection during in vitro seed inoculation bioassays. Transgenic peanut plants evaluated for resistance against late leaf spot (LLS) and rust using detached leaf assays showed longer incubation, latent period and lower infection frequencies when compared to their non-transformed counterparts. A significant negative correlation existed between the chitinase activity and the frequency of infection to the three tested pathogens. Three progenies from two transgenic events displayed significantly higher disease resistance for LLS, rust and A. flavus infection and are being advanced for further evaluations under confined field conditions to confirm as sources to develop peanut varieties with enhanced resistance to these fungal pathogens.
TL;DR: Evidence is provided for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain’s antifungal activity and insecticidal activity.
Abstract: A novel strain exhibiting entomopathogenic and chitinolytic activity was isolated from mangrove marsh soil in India. The isolate was identified as Brevibacillus laterosporus by phenotypic characterization and 16S rRNA sequencing and designated Lak1210. When grown in the presence of colloidal chitin as the sole carbon source, the isolate produced extracellular chitinases. Chitinase activity was inhibited by allosamidin indicating that the enzymes belong to the family 18 chitinases. The chitinases were purified by ammonium sulfate precipitation followed by chitin affinity chromatography yielding chitinases and chitinase fragments with 90, 75, 70, 55, 45, and 25 kDa masses. Mass spectrometric analyses of tryptic fragments showed that these fragments belong to two distinct chitinases that are almost identical to two putative chitinases, a 89.6-kDa four-domain chitodextrinase and a 69.4-kDa two-domain enzyme called ChiA1, that are encoded on the recently sequenced genome of B. laterosporus LMG15441. The chitinase mixture showed two pH optima, at 6.0 and 8.0, and an optimum temperature of 70 °C. The enzymes exhibited antifungal activity against the phytopathogenic fungus Fusarium equiseti. Insect toxicity bioassays with larvae of diamondback moths (Plutella xylostella), showed that addition of chitinases reduced the time to reach 50 % mortality upon infection with non-induced B. laterosporus from 3.3 to 2.1 days. This study provides evidence for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain’s antifungal activity and insecticidal activity.
TL;DR: Pretreated chitin substrates were pretreated with sonication and steam explosion prior to enzymatic reaction, and Structural changes were observed with steam explosion with 11.28% reduction of the crystallinity index attained with the lowest chit in/water ratio.
TL;DR: The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitIn in the manufacture of food and/or medicine.
Abstract: Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.
TL;DR: The results of this research prove that the substances which are metabolised by S. rimosus the most efficiently are chitosan and the shrimp shell waste, the finest inducer of chitinase synthesis.
TL;DR: The actinomycetes Streptomyces violaceusniger showed strong antagonistic activity against various tested wood rotting fungi and an extracellular chitinase, produced by antagonistic S. violaceUSniger MTCC 3959, was purified as follows: ammonium sulfate precipitation, chit in affinity and chromatographic separation of Q Sepharose.
Abstract: The actinomycetes Streptomyces violaceusniger showed strong antagonistic activity against various tested wood rotting fungi. An extracellular chitinase, produced by antagonistic S. violaceusniger MTCC 3959, was purified as follows: ammonium sulfate precipitation, chitin affinity and chromatographic separation of Q Sepharose. The molecular mass of the purified chitinase was estimated as 56.5 kDa by SDS-PAGE. Chitinase was optimally active at pH of 5.0 and 50 °C. It retained almost 100% activity at pH 5.0 and also had high thermal tolerance at 50 °C. Enzyme activity was inhibited by Hg(2+) and Ag(+) cations, but was neither substantially inhibited by K(+) cation nor by chelating agent EDTA. The apparent Km and Vmax at 37 °C were 0.1426 mM and 6.6 U/mg, respectively using pNP-(GlcNAc)2 as substrate. The 56.5 kDa chitinase of strain MTCC 3959 represented an exo-type activity. The purified chitinase was further identified by MALDI-TOF. The results of peptide mass fingerprinting showed that 10 tryptic peptides of the chitinase were identical to the chitinase C from Streptomyces albus J1074 (GenBank Accession No. gi|239982330). The sequence of N-terminal amino acid (AA) of the chitinase was determined to be G-D-G-T-G-P-G-P-G-P.
TL;DR: Stenotrophomonas maltophilia chitinase genes were cloned and expressed as soluble proteins of 70.5 and 41.6 kDa in Escherichia coli and hydrolytic activity on chitooligosaccharides indicated that StmChiA was an endo-acting enzyme releasing chitobiose and StmchiB was both exo/endo- acting enzyme with the release of GlcNAc as the final product.
TL;DR: A reconsideration in terms of understanding the roles of chitinolytic enzymes in applications, e.g. host–pathogen interaction for biocontrol, different mechanisms of ch itin degradation, and identification of new enzymes with varying specificities may make them more useful in a variety of commercial processes in the near future.
Abstract: Chitin, its deacetylated form, chitosan and chitinolytic enzymes viz. endo-chitinase, N-acetylglucosaminidase, chitosanase, chitin deacetylase (CDA) are gaining importance for their biotechnological applications. Presently, chitin degrading enzymes constitute high-cost low-volume products in human health care and associated research. Indeed chitinases and CDA-chitosanase complex possesss tremendous potential in agriculture to control plant pathogenic fungi and insects. The success in exploring chitinases especially for agriculture, i.e. as a high-volume low-cost product, depends on the availability of highly active preparations with a reasonable cost. Therefore, a reconsideration in terms of understanding the roles of chitinolytic enzymes in applications, e.g. host-pathogen interaction for biocontrol, different mechanisms of chitin degradation, and identification of new enzymes with varying specificities, may make them more useful in a variety of commercial processes in the near future. The possible issues and challenges encountered in the translation of proof of concept into a commercial product will be appraised in this review.
TL;DR: Results may serve as a base line data for enhancing the chitinolytic potential of bacterial antagonists for bio‐management of chickpea pathogens.
Abstract: Chitinase producing strain B-CM18 was isolated from chickpea rhizosphere and identified as Lysinibacillus fusiformis B-CM18. It showed in vitro antifungal activity against a wide range of fungal plant pathogens and was found to produce several PGPR activities. Further, a multivariate response surface methodology was used to evaluate the effects of different factors on chitinolytic activity and optimizing enzyme production. A central composite design was employed to achieve the highest chitinase production at optimum values of the process variables, viz., temperature (20–45 °C), sodium chloride (2–7%), starch (0.1–1%) and yeast extract (0.1–1%), added in the minimal medium supplemented with colloidal chitin (1–10%; w:w). The fit of the model (R2 = 0.5692) was found to be significant. The production medium to achieve the highest chitinase production (101 U ml−1) was composed of the minimal medium composed of chitin (6.09%), NaCl (4.5%), starch (0.55%) and yeast extract (0.55%) with temperature (32.5 °C). The results show that the optimization strategy led to an increase in chitinase production by 56.1-fold. The molecular mass of the chitinase was estimated to be 20 kDa by anion exchange and gel filtration chromatography. Further, purified chitinase showed strong antifungal activity against test pathogens. Overall, these results may serve as a base line data for enhancing the chitinolytic potential of bacterial antagonists for bio-management of chickpea pathogens.
TL;DR: A highly chitinolytic strain Penicillium ochrochloron MTCC 517, which gives N-acetylglucosamine as the main hydrolyzate after hydrolysis of colloidal chitin, seems to be a promising biocontrol agent.
TL;DR: These results were the first bioethanol production from GlcNAc and chitin substrates by native organisms, and suggest that these Mucor strains have great potential for the simultaneous saccharification and fermentation (SSF) of chit in biomass.
TL;DR: The application of chiIO8 treatment was also observed to improve the germinative energy and the enhancing effects of the chiio8 on disinfectant properties against seed‐borne diseases.
Abstract: Aims
To investigate the distribution of chitinase IO8 in Bacillus cereus strains, the enhancing effects of the chitinase-producing B. cereus strains on biocontrol potential by dual culture assay and in vivo assay against Botrytis cinerea and also the enhancing effects of the chiIO8 on disinfectant properties against seed-borne diseases. Moreover, the application of chiIO8 treatment was also observed to improve the germinative energy.
Methods and Results
The purification steps included ammonium sulfate precipitation, with columns of DEAE-Sepharose anion-exchange chromatography and Sephacryl S-400 high-resolution gel chromatography. The method gave a 5·8-fold increase in the specific activity and had a yield of 17%. The molecular weight of the partially purified chitinase chiIO8 was found to be around 30 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and optimal temperature of the partially purified chitinase were pH 6·5 and 65°C, respectively. The thermostable chitinase still retained the activity after incubation for 100 min at 65°C, and it was increased about 1·25 times than that of the control (before heating) when the enzyme solution heated at 65°C for 60 min. The partially purified chitinase chiIO8 displays a wide inhibitory spectrum towards all phytopathogenic fungi tested. chiIO8 also exhibited effective disinfectant properties against seed-borne diseases.
Conclusion
The present investigation emphasizes the potential of chitinase-producing micro-organism as promising biocontrol agents of fungal plant pathogens with chitinous cell wall. The novel chitinase chiIO8 proved an efficient, environmentally safe and user-friendly solution.
Significance and Impact of the Study
This is the first investigation devoted exclusively to analyse the distribution of chitinase in B. cereus. It infers that the chitinase produced by B. cereus might play a role in the activity of the biopesticide.
TL;DR: It is demonstrated that a Listeria monocytogenes-secreted chitinase has distinct functional roles that include chit in hydrolysis and suppression of host innate immunity.
Abstract: Environmental pathogens survive and replicate within the outside environment while maintaining the capacity to infect mammalian hosts. For some microorganisms, mammalian infection may be a relatively rare event. Understanding how environmental pathogens retain their ability to cause disease may provide insight into environmental reservoirs of disease and emerging infections. Listeria monocytogenes survives as a saprophyte in soil but is capable of causing serious invasive disease in susceptible individuals. The bacterium secretes virulence factors that promote cell invasion, bacterial replication, and cell-to-cell spread. Recently, an L. monocytogenes chitinase (ChiA) was shown to enhance bacterial infection in mice. Given that mammals do not synthesize chitin, the function of ChiA within infected animals was not clear. Here we have demonstrated that ChiA enhances L. monocytogenes survival in vivo through the suppression of host innate immunity. L. monocytogenes Δ chiA mutants were fully capable of establishing bacterial replication within target organs during the first 48 h of infection. By 72 to 96 h postinfection, however, numbers of Δ chiA bacteria diminished, indicative of an effective immune response to contain infection. The Δ chiA -associated virulence defect could be complemented in trans by wild-type L. monocytogenes, suggesting that secreted ChiA altered a target that resulted in a more permissive host environment for bacterial replication. ChiA secretion resulted in a dramatic decrease in inducible nitric oxide synthase (iNOS) expression, and Δ chiA mutant virulence was restored in NOS2 − / − mice lacking iNOS. This work is the first to demonstrate modulation of a specific host innate immune response by a bacterial chitinase. IMPORTANCE Bacterial chitinases have traditionally been viewed as enzymes that either hydrolyze chitin as a food source or serve as a defense mechanism against organisms containing structural chitin (such as fungi). Recent evidence indicates that bacterial chitinases and chitin-binding proteins contribute to pathogenesis, primarily via bacterial adherence to chitin-like molecules present on the surface of mammalian cells. In contrast, mammalian chitinases have been linked to immunity via inflammatory immune responses that occur outside the context of infection, and since mammals do not produce chitin, the targets of these mammalian chitinases have remained elusive. This work demonstrates that a Listeria monocytogenes-secreted chitinase has distinct functional roles that include chitin hydrolysis and suppression of host innate immunity. The established link between chitinase and the inhibition of host inducible nitric oxide synthase (iNOS) expression may help clarify the thus far elusive relationship observed between mammalian chitinase enzymes and host inflammatory responses occurring in the absence of infection.
TL;DR: European vespertilionid bat species have acidic mammalian chitinase that is produced in the gastric glands of the stomach, and the gastrointestinal tracts of insectivorous bat species evolved an enzymatic adaptation to their diet.
Abstract: The gastrointestinal tract of animals is adapted to their primary source of food to optimize resource use and energy intake. Temperate bat species mainly feed on arthropods. These contain the energy-rich carbohydrate chitin, which is indigestible for the endogenous enzymes of a typical mammalian gastrointestinal tract. However, the gastrointestinal tract of bat species should be adapted to their diet and be able to digest chitin. We hypothesized that (i) European vespertilionid bat species have the digestive enzyme chitinase and that (ii) the chitinolytic activity is located in the intestine, as has been found for North American bat species. The gastrointestinal tracts of seven bat species (Pipistrellus pipistrellus, Plecotus auritus, Myotis bechsteinii, Myotis nattereri, Myotis daubentonii, Myotis myotis, and Nyctalus leisleri) were tested for chitinolytic activity by diffusion assay. Gastrointestinal tracts of P. pipistrellus, P. auritus, M. nattereri, M. myotis, and N. leisleri were examined for acidic mammalian chitinase by western blot analysis. Tissue sections of the gastrointestinal tract of P. pipistrellus were immunohistochemically analyzed to locate the acidic mammalian chitinase. Chitinolytic activity was detected in the stomachs of all bat species. Western blot analysis confirmed the acidic mammalian chitinase in stomach samples. Immunohistochemistry of the P. pipistrellus gastrointestinal tract indicated that acidic mammalian chitinase is located in the stomach chief cells at the base of the gastric glands. In conclusion, European vespertilionid bat species have acidic mammalian chitinase that is produced in the gastric glands of the stomach. Therefore, the gastrointestinal tracts of insectivorous bat species evolved an enzymatic adaptation to their diet.
TL;DR: The 25-kDa protein was shown to also degrade chitin, suggesting that isolate 76T-2 has the ability to degrade both PCL and chitIn, indicating the presence of extracellular PCL depolymerases.
Abstract: A thermophilic Streptomyces thermoviolaceus subsp. thermoviolaceus isolate 76T-2 that can degrade poly(e-caprolactone) (PCL) was isolated from soil in Taiwan. Isolate 76T-2 grew well in urea fructose oatmeal medium and exhibited clear zones on agar plates containing PCL, indicating the presence of extracellular PCL depolymerases. The PCL powder present in culture medium was completely degraded within 6 h of culture at 45°C. Two PCL-degrading enzymes were purified to homogeneity from the culture supernatant. The molecular weights of these two enzymes were estimated to be 25 kDa and 55 kDa, respectively. A portion of the N-terminal region of the 25-kDa protein was determined, and the sequence Ala-Asn-Phe-Val-Val-Ser-Glu-Ala thus obtained was identical to that of A64-A71 of the Chi25 chitinase of Streptomyces thermoviolaceus OPC-520. The 25-kDa protein was shown to also degrade chitin, suggesting that isolate 76T-2 has the ability to degrade both PCL and chitin.
TL;DR: The gene expression patterns suggest that chitinase and β-N-acetylglucosaminidase may be responsible for the degradation of larval cuticles, whereas chitIn deacetylase may help to degrade the pupal counterparts, and 20E-induced nuclear receptors seem to be important factors regulating chit in metabolic enzymes during the cuticle remodeling.
TL;DR: Studying the contribution of monocomponent chitinases and the lytic polysaccharide monooxygenase from Serratia marcescens on depolymerization of α-chitin substrates with varying particle size and crystallinity revealed clear synergies for all substrates tested.
Abstract: Industrial depolymerization of chitinous biomass generally requires numerous steps and the use of deleterious substances. Enzymatic methods provide an alternative, but fundamental knowledge that could direct potential development of industrial enzyme cocktails is scarce. We have studied the contribution of monocomponent chitinases (ChiA, -B, and -C) and the lytic polysaccharide monooxygenase (LPMO) from Serratia marcescens on depolymerization of α-chitin substrates with varying particle size and crystallinity that were generated using a converge mill. For all chitinases activity was positively correlated to a decline in particle size and crystallinity. Especially ChiC, the only nonprocessive endochitinase from the S. marcescens chitinolytic machinery, benefited from mechanical pretreatment. Combining the chitinases revealed clear synergies for all substrates tested. CBP21, the chitin-active LPMO from S. marcescens, increased solubilization of substrates with high degrees of crystallinity when combined with each of the three chitinases, but this synergy was reduced upon decline in crystallinity.
TL;DR: OfCht5, the glycoside hydrolase family 18 chitinase from the widespread lepidopteran pest Ostrinia furnacalis, was cloned, expressed in the yeast Pichia pastoris and biochemically characterized in an attempt to facilitate both pest control and biomaterial preparation.
Abstract: Chitinase catalyzes β-1,4-glycosidic linkages in chitin and has attracted re- search interest due to it being a potential pesticide target and an enzymatic tool for preparation of N-acetyl-β-D-glucosamine. An individual insect contains multiple genes encodingchitinases,whichvaryindomainarchitectures,expressionpatterns,physiological roles and biochemical properties. Herein, OfCht5, the glycoside hydrolase family 18 chiti- nase from the widespread lepidopteran pest Ostrinia furnacalis, was cloned, expressed in the yeast Pichia pastoris and biochemically characterized in an attempt to facilitate both pest control and biomaterial preparation. Complementary DNA sequence analysis indicated that OfCHT5 consisted of an open reading frame of 1665-bp nucleotides. Phy- logenic analysis suggested OfCht5 belongs to the Group I insect chitinases. Expression of OfCht5 in Pichia pastoris resulted in highest specific activity after 120 h of induction with methanol. Through two steps of purification, consisting of ammonium sulfate pre- cipitation and metal chelating chromatography, about 7 mg of the recombinant OfCht5 was purified to homogeneity from 1 L culture supernatant. OfCht5 effectively converted colloidal chitin into chitobiose, but had relatively low activity toward α-chitin. When chi- tooligosaccharides ((GlcNAc)n, n = 3-6) were used as substrates, OfCht5 was observed to possess the highest catalytic efficiency parameter toward (GlcNAc)4 and predominan- tely hydrolyzed the second glycosidic bond from the non-reducing end. Together with β-N-acetyl-D-hexosaminidase OfHex1, OfCht5 achieved its highest efficiency in chitin degradation that yielded N-acetyl-β-D-glucosamine, a valuable pharmacological reagent and food supplement, within a molar concentration ratio of OfCht5 versus OfHex1 in the range of 9:1-15:1. This work provides an alternative to existing preparation of chitinase for pesticides and other applications.