Chitinolytic Enzyme Activity Research Articles

Diversity of Culturable Chitinolytic Bacteria from Rhizospheres of Agronomic Plants in Japan

A total of 100 isolates of chitinolytic bacteria were obtained from the rhizospheres of various agronomic plants, and the 16S rRNA gene sequences of these isolates were determined. Phylogenetic analyses revealed that 81 isolates belonged to the classes Betaproteobacteria (39 isolates) and Gammaproteobacteria (42 isolates). Of the remaining 19 isolates, 16 belonged to the phylum Firmicutes. Clustering analysis identified 6 and 3 operational taxonomic units (OTUs) in Gammaproteobacteria and Betaproteobacteria, respectively, at the genus level. The majority of chitinolytic bacteria in Gammaproteobacteria belonged to the genera Serratia, Stenotrophomonas, and Lysobacter (14, 15, and 7 isolates, respectively) while those in Betaproteobacteria belonged to the genus Mitsuaria (37 isolates). The 16 isolates placed in Firmicutes belonged to 2 genera, Paenibacillus and Bacillus (8 isolates each). The isolates in the remaining OTUs belonged to the genera Erwinia, Aeromonas, Pseudomonas, Achromobacter, Flavobacterium, and Microbacterium, in less abundance. These results showed a wide distribution of culturable chitinolytic bacteria in the rhizospheres of various agronomic plants. Considering the potential antagonistic activity of chitinolytic enzymes against phytopathogenic fungi, which is exhibited by fungal cell wall degradation, the above-mentioned native chitinolytic bacteria in rhizospheres could potentially be utilized for the biological control of soil-borne phytopathogenic fungi.

The Chitinolytic Activities of Streptomyces sp. TH-11

Chitin is an abundant biopolymer composed of units of N-acetyl-D-glucosamine linked by β-1,4 glycosidic bonds. Chitin is the main component of the shells of mollusks, the cell wall of fungi and yeast and of the exoskeleton of crustaceans and insects. The degradation of chitin is catalyzed by chitinases that occur in a wide range of organisms. Among them, the chitinases from microorganisms are extremely important for the degradation and recycling of the carbon and nitrogen trapped in the large amount of insoluble chitin in nature. Streptomyces sp. TH-11 was isolated from the sediment of the Tou-Chien River, Taiwan. The chitinolytic enzyme activities were detected using a rapid in-gel detection method from the cell-free preparation of the culture medium of TH-11. The chitinolytic enzyme activity during prolonged liquid culturing was also analyzed by direct measurement of the chitin consumption. Decomposition of the exoskeleton of shrimps was demonstrated using electron microscopy and atomic force microscopy.

Production of extracellular enzymes and degradation of biopolymers by saprotrophic microfungi from the upper layers of forest soil

Production of extracellular enzymes participating in the degradation of biopolymers was studied in 29 strains of nonbasidiomycetous microfungi isolated from Quercus petraea forest soil based on the frequency of occurrence. Most of the isolates were ascomycetes and belonged to the genera Acremonium, Alternaria, Cladosporium, Geomyces, Hypocrea, Myrothecium, Ochrocladosporium, and Penicillium (18 isolates), and two isolates were zygomycetes. Only six isolates showed phenol oxidation activity which was low and none of the strains were able to degrade humic acids. Approximately half of the strains were able to degrade cellulose and all but six degraded chitin. Most strains produced significant amounts of the cellulolytic enzymes cellobiohydrolase and β-glucosidase and the chitinolytic enzymes chitinase, chitobiosidase, and N-acetylglucosaminidase. The highest cellulase activities were found in Penicillium strains, and the highest activity of chitinolytic enzymes was found in Acremonium sp. The production of the hemicellulose-degrading enzymes α-galactosidase, β-galactosidase, and α-mannosidase was mostly low. The microfungal strains were able to produce significant growth on a range of 41–87, out of 95 simple C-containing substrates tested in a Biolog™ assay, monosaccharides being for all strains the most rapidly metabolized C-sources. Comparison with saprotrophic basidiomycetes from the same environment showed that microfungi have similar cellulolytic capabilities and higher chitinase activities which testifies for their active role in the decomposition of both lignocellulose and dead fungal biomass, important pools of soil carbon.

Differential expression of maize chitinases in the presence or absence of Trichoderma harzianum strain T22 and indications of a novel exo- endo-heterodimeric chitinase activity.

BackgroundThe interaction of plants with endophytic symbiotic fungi in the genus Trichoderma alters the plant proteome and transcriptome and results in enhanced plant growth and resistance to diseases. In a previous study, we identified the numerous chitinolytic enzyme families and individual enzymes in maize which are implicated in plant disease resistance and other plant responses.ResultsWe examined the differential expression of the entire suite of chitinolytic enzymes in maize plants in the presence and absence of T. harzianum. Expression of these enzymes revealed a band of chitinolytic enzyme activity that had greater mass than any known chitinase. This study reports the characterization of this large protein. It was found to be a heretofore undiscovered heterodimer between an exo- and an endo-enzyme, and the endo portion differed between plants colonized with T. harzianum and those grown in its absence and between shoots and roots. The heterodimeric enzymes from shoots in the presence and absence of T. harzianum were purified and characterized. The dimeric enzyme from Trichoderma-inoculated plants had higher specific activity and greater ability to inhibit fungal growth than those from control plants. The activity of specific chitinolytic enzymes was higher in plants grown from Trichoderma treated seeds than in control plants.ConclusionsThis is the first report of a dimer between endo- and exochitinase. The endochitinase component of the dimer changed post Trichoderma inoculation. The dimer originating from Trichoderma inoculated plants had a higher antifungal activity than the comparable enzyme from control plants.

Genome-wide identification, expression and chromosomal location of the genes encoding chitinolytic enzymes in Zea mays

Chitinolytic enzymes are important pathogenesis and stress related proteins. We identified 27 putative genes encoding endochitinases in the maize genome via in silico techniques and four exochitinases. Only seven of the endochitinases and segments of the exochitinases were heretofore known. The endochitinases included members of family 19 chitinases (classes I-IV of PR3, II of PR4) and members of family 18 chitinases (class III of PR8). Some similar enzymes were detected on adjacent regions of the same chromosome, and seem to result from duplication events. Most of the genes expressed were identified from EST libraries from plants exposed to biotic or abiotic stresses but also from libraries from tissues not exposed to stresses. We isolated proteins from seedlings of maize in the presence or absence of the symbiotic root colonizing fungus Trichoderma harzianum strain T22, and analyzed the activity of chitinolytic enzymes using an in-gel activity assay. The activity bands were identified by LC/MS/MS using the database from our in silico study. The identities of the enzymes changed depending on whether or not T22 was present. One activity band of about 95 kDa appeared to be a heterodimer between an exochitinase and any of several different endochitinases. The identity of the endochitinase component appeared to be dependent upon treatment.

Glycolytic and chitinolytic activities of Phlebotomus papatasi (Diptera: Psychodidae) from diverse ecological habitats

The sand fly Phlebotomus papatasi Scopoli, 1786, the vector of Leishmania major Yakimoff et Schokhor, 1914, is found in desert areas where sugars are scarce but also in habitats that abound in sugar sources. The sand flies require sugar meals from plant sources for their energy requirements and to hydrolyze these complex sugars, they need a repertoire of glycosidases. We presumed that there are differences in the levels of glycosidase activities in flies from such habitats and also assumed that they may be instrumental in modulating the flies' susceptibility to L. major infections. Phlebotomus papatasi originating from diverse ecological habitats ranging from an oasis to desert sites were colonized. They were analyzed for weight changes and glycosidase activities before and after feeding on 1M sucrose solution. Oasis flies were smaller than desert flies but took larger sugar meals. Homogenates of these flies hydrolyzed 16 synthetic and 2 natural glycoside substrates to varying degrees. The arid-region flies tended to produce more glycosidase activity than those originating in sugar-rich environments, especially sucrase, alpha- and beta-glucosidase, aalpha-fucosidase, alpha-mannosidase, and alpha- and beta-N-acetylgalactosaminidase. However, chitinolytic enzyme activities and particularly the beta-N-acetylhexosaminidase activity of oasis flies were higher than other flies tested. In comparing the desert flies, there were also significant differences in glycolytic enzyme activities between the spring-line (flowering season) of flies and the autumn-line (end of dry season) flies. A range of saccharide inhibitors was tested to demonstrate the specificity of the enzymes.

The Phylogenetically Conserved Molluscan Chitinase-like Protein 1 (Cg-Clp1), Homologue of Human HC-gp39, Stimulates Proliferation and Regulates Synthesis of Extracellular Matrix Components of Mammalian Chondrocytes

Members of chitinase-like proteins (CLPs) have attracted much attention because of their ability to promote cell proliferation in insects (imaginal disc growth factors) and mammals (YKL-40). To gain insights into the molecular processes underlying the physiological control of growth and development in Lophotrochozoa, we report here the cloning and biochemical characterization of the first Lophotrochozoan CLP from the oyster Crassostrea gigas (Cg-Clp1). Gene expression profiles monitored by real time quantitative reverse transcription-PCR in different adult tissues and during development support the involvement of this protein in the control of growth and development in C. gigas. Recombinant Cg-Clp1 demonstrates a strong affinity for chitin but no chitinolytic activity, as was described for the HC-gp39 mammalian homolog. Furthermore, transient expression of Cg-Clp1 in primary cultures of rabbit articular chondrocytes as well as the use of both purified recombinant protein and conditioned medium from Cg-Clp1-expressing rabbit articular chondrocytes established that Cg-Clp1 stimulates cell proliferation and regulates extracellular matrix component synthesis, showing for the first time a possible involvement of a CLP on type II collagen synthesis regulation. These observations together with the fact that Cg-Clp1 gene organization strongly resembles that of its mammalian homologues argue for an early evolutionary origin and a high conservation of this class of proteins at both the structural and functional levels.

Enzyme activity assays for nutrient solutions from closed irrigation systems

Exo-enzymes degrading cell wall components of fungal pathogens may play a role in the biological control of plant diseases in hydroponic systems. Therefore, protease, cellulase and chitinase are of special interest. The main objective of this work was the development of enzyme assays suitable for the conditions found in nutrient solutions. Furthermore, inducibility of enzyme activities by amendment of organic substrates to nutrient solutions and the effect of storage on enzyme activities in nutrient solutions were investigated. Enzyme tests based on the rate of release of soluble dye-labeled fragments of chromogenic substrates were adapted for nutrient solutions and were evaluated as suitable for the purpose. Using Hide-Remazol Brilliant Blue R as substrate for protein degrading enzymes, a stable activity of protease for up to 16 h was found. In order to detect the activity of chitinolytic enzymes, Carboxymethyl-Chitin Remazol Brilliant Violet was utilized as substrate. Results indicated good performance of the test, although chitinase activity was not observed in the beginning but later in the crop season. Furthermore, an approach to detect cellulase activity using Carboxymethyl-Cellulose Remazol Brilliant Blue as substrate yielded satisfying results. Amendment of nutrient solutions with chitin and cellulose (1%) incubated for 3 days led to significantly increased activity of chitinase and cellulase, respectively. For all of the studied enzymes, storage at +2 °C did not affect the activity for at least 1 week. In contrast, storage of samples of nutrient solution at room temperature led to a rapid decrease in activity of chitinase and cellulase, whereas protease activity was negatively affected by freezing (−26 °C) the nutrient solution.

Synergistic Antifungal Activity of Chitinolytic Enzymes and Prodigiosin Produced by Biocontrol Bacterium, Serratia marcescens Strain B2 against Gray Mold Pathogen, Botrytis cinerea

strain B2 is an effective biocontrol agent against gray mold of cyclamen, caused by Botrytis cinerea Persoon. Strain B2 has strong antifungal activity against B. cinerea in vitro. The culture filtrate was found to contain different types of chitinolytic enzyme activity, including endochitinase and chitobiase activity. Four chitinolytic enzymes were detected among the extracellular proteins of strain B2. Two major enzymes, a 58-kDa endochitinase and a 98-kDa chitobiase, were purified by chromatography and electrofocusing, respectively. Both enzymes inhibited spore germination of B. cinerea. And a red pigment, prodigiosin, extracted and purified from the bacterial cells, also inhibited spore germination. When prodigiosin and the chitinolytic enzymes were applied in concert, a synergistic inhibitory effect was observed. S. marcescens strain B2 has multiple modes of action against the pathogen.

Hydrolytic enzyme activity of Paenibacillus sp. strain B2 and effects of the antagonistic bacterium on cell integrity of two soil-borne pathogenic fungi

Paenibacillus sp. strain B2, isolated from the mycorrhizosphere of Sorghum bicolor and having an antagonistic activity towards soil-borne fungal pathogens, possessed extracellular cellulolytic, proteolytic, chitinolytic and pectinolytic enzyme activities. The eventual role of these lytic enzymes in cellular interactions between Paenibacillus sp. strain B2 and Phytophthora parasitica and Fusarium oxysporum was investigated by electron microscopy and molecular cytology. Electron microscopic observations showed that the presence of Paenibacillus sp. strain B2 resulted in disorganisation of cell walls and/or cell contents of P. parasitica and F. oxysporum. However, when P. parasitica was treated with commercial purified cellulase, protease, chitinase and pectinase, only protease had an inhibitory effect on mycelial growth. It is proposed that the inhibitory effect of Paenibacillus sp. strain B2 on the growth of soil-borne fungal pathogens is probably derived from more than one mechanism.

Chitinase Gene Expression during Mycoparasitic Interaction ofTrichoderma harzianumwith Its Host

Zeilinger, S., Galhaup, C., Payer, K., Woo, S. L., Mach, R. L., Fekete, C., Lorito, M., and Kubicek, C. P. 1999. Chitinase gene expression during mycoparasitic interaction ofTrichoderma harzianumwith its host.Fungal Genetics and Biology26,131–140. For monitoring chitinase expression during mycoparasitism ofTrichoderma harzianum in situ,we constructed strains containing fusions of green fluorescent protein (GFP) to the 5′-regulatory sequences of theT. harzianum nag1(N-acetyl-β-d-glucosaminidase-encoding) andech42(42-kDa endochitinase-encoding) genes. Confronting these strains withRhizoctonia solaniled to induction of gene expression before (ech42) or after (nag1) physical contact. A 12-kDa cut-off membrane separating the two fungi abolishedech42expression, indicating that macromolecules are involved in its precontact activation. Noech42expression was triggered by culture filtrates ofR. solanior by placingT. harzianumonto plates previously colonized byR. solani.Instead, high expression occurred upon incubation ofT. harzianumwith the supernatant ofR. solanicell walls digested with culture filtrates or purified endochitinase 42 (CHIT42, encoded byech42) fromT. harzianum.The chitinase inhibitor allosamidin blockedech42expression and reduced inhibition ofR. solanigrowth during confrontation. The results indicate thatech42is expressed before contact ofT. harzianumwithR. solaniand its induction is triggered by soluble chitooligosaccharides produced by constitutive activity of CHIT42 and/or other chitinolytic enzymes.

Chitinolytic enzyme activity of Penicillium janthinellum P9 in bench-top bioreactor

The chitinolytic activity of Penicillium janthinellum P9 was studied in shaken cultures and in a 3- l bench-top bioreactor by varying culture conditions such as initial medium pH, growth temperature, stirrer speed and aeration site. In shaken flasks, the highest levels of enzyme activity (468 and 483 U· l −1) were obtained at a growth temperature of 24°C and at an initial medium pH of 4.0, respectively. In the bioreactor, both agitation and aeration significantly influenced the enzyme production: the highest level of enzyme activity (497 U· l −1) was obtained at an impeller speed of 500 rpm and an aeration rate of 1.5 vvm. Culturing P. janthinellum P9 under optimised conditions led to an increase in the enzyme activity of ca. 65% (686 U· l −1 as compared to the 415 U· l −1 obtained under the initial culture conditions).

Location of chitinolytic enzymes in protoplasts and whole cells of the entomopathogenic fungus Metarhizium anisopliae

Metarhizium anisopliae produces chitinolytic enzymes which have been implicated in digestion of the cuticles of the host insect during the infection process. In this study, enzymatically produced fungal protoplasts and whole mycelium have been used to study the cellular location and regulation of these enzymes. Intact mycelium was not significantly induced by soluble carboxymethylated (alkaline) chitin in the production of chitinolytic enzymes. In contrast, enzymatically produced protoplasts, of M. anisopliae were highly inducible for these enzymes. At 24 h incubation, chitin induced protoplasts secreted into the medium 76% of total N -acetylglucosaminidase activity, whereas less than 10% of chitinase activity was secreted. At 48 h incubation, however, N -acetylglucosaminidase secretion had dropped to 34% of total activity, while the proportion of chitinase activity secreted by protoplasts continued to rise. By 48 h, the majority of chitinolytic enzyme activity had become cell-bound in both protoplast preparations and whole cells, and in protoplasts, this activity was mainly located in the membrane/developing wall fraction. The influence of the cell wall in the regulation and secretion of these enzymes is discussed.

Chitinolytic Enzyme Activities in the Hepatopancreas, Tail Fan and Hemolymph of Kuruma Prawn <i>Penaeus japonicus</i> during the Molt Cycle

Chitinolytic Enzyme Activities in the Hepatopancreas, Tail Fan and Hemolymph of Kuruma Prawn <i>Penaeus japonicus</i> during the Molt Cycle

Interference of the insect growth regulator methoprene in the process of larval‐pupal differentiation

AbstractStudies with epidermal cells of a number of insects showed that a sequence of physiological events takes place prior to and during the process of commitment of the epidermal cells from the larval to the pupal stage. At the onset of metamorphosis RNA synthesis, including that of RNA, was suppressed for about 30 h just prior to the wandering stage. At the completion of the wandering stage, pupally committed epidermal cells synthesize a new type of RNA termed hetero‐disperse nuclear RNA (hnRNA). It is suggested that this RNA species could play a role in the permanent shutting‐off of larval‐specific genes. Methoprene, a juvenile hormone analogue (JHA), delayed the larval‐pupal transformation and prevented these changes in RNA synthesis when applied prior to the appearance of the hnRNA, but had no effect when applied after this period. Treatment with the JHA in the early last larval instar prevented not only the normal rise in ecdysteroid titer, but also the increase in the activity of chitinolytic enzymes. Treatment of young pupae had no effect on either the ecdysteroids titer or on enzyme activity. A causal relationship between molting hormone titer and the activity of chitinolytic enzymes is proposed. © 1993 Wiley‐Liss, Inc.

Antifungal, Synergistic Interaction Between Chitinolytic Enzymes fromTrichoderma harzianumandEnterobacter cloacae

Biocontrol strains from the genera Enterobacter and Pseudomonas and two chitinolytic enzymes from Trichoderma harzianum isolate P1 were combined and tested for antifungal activity in bioassays. Inhibitory effects on spore germination and germ tube elongation of Botrytis cinerea, Fusarium solani, and Uncinula necator were synergistically increased by mixing fungal enzymes and cells of Enterobacter cloacae but not of Pseudomonas spp. Culture filtrate of E. cloacae contained antifungal compounds and produced moderate levels of inhibition, either in plate assays or in bioassays conducted in potato-dextrose broth. However, the combination of bacterial culture filtrate with fungal chitinolytic enzymes generated only an additive response, indicating that the presence of bacterial cells was required for a synergistic effect. Chitinolytic enzyme activity in the presence of chitinous substrates enhanced the growth of E. cloacae and readily restored the ability of bacterial cells to bind to hyphae of the pathogens despite high concentrations of D-glucose or sucrose in the medium. The results of this study suggest that transgenic bacteria, capable of binding to fungal cell walls and expressing fungal genes encoding cell wall-degrading enzymes, may be powerful biocontrol agents

Endochitinase fromGliocladium virens: Isolation, Characterization, and Synergistic Antifungal Activity in Combination with Gliotoxin

The culture filtrate from the biocontrol agent Gliocladium virens strain 41 grown in chitin-containing medium was strongly inhibitory to mycelial growth of different plant-pathogenic fungi. The antibiotic gliotoxin was isolated from the culture liquid. The culture filtrate also contained different types of chitinolytic enzyme activities, including endochitinase, chitin 1,4β-chitobiosidase, and glucan N-acetyl-β-D-glucosaminidase, as well as glucan 1,3-β-glucosidase activity. An endochitinase was purified to homogeneity []

Changes in Chitin and N-Acetylglucosamine Contents and Chitinolytic Enzyme Activities during te Growth of Flammulina velutipes Fruit-bodies.

We studied the changes in chitin and N-acetylglucosamine(GlcNAc)contents and activities of chitinase and β-N-acetylglucosaminidase(β-GlcNAcase)during the fruit-body development of Flammulina uelutipes (Enokitake) which is one of the important edible mushrooms in Japan. The strain used (IFO7777) formed a matured fruit-body in liquid culture in 25 days. The chitin components of the fungus were 1.0-1.5%in dry mycelia and 2.0-4.0% in dry fruit-bodies, though both components were lower than that of the other fungi of basidiomycotina. The former components linearly increased during the mycelial growth, but when the fruit-body formation began, the increase was considerably suppressed. On the other hand, the amounts of GlcNAc in the culture filtrate and mycelia suddenly decreased at the stage of rapid growth of the fruit-body. It is, therefore, suggested that GlcNAc as a substrate is supplied from them for the synthesis of chitin in the fruit-body. Chitinase activity in mycelia increased at the growing stage of vegetative mycelia, but it was suppressed with the beginning of rapid development of fruit-body. However its high activity continued until the maturation of the fruit-body. The activity in the culture filtrate continuously increased until the maturation of the fruit-body. β-GlcNAcase activity showed a diererent pattern from that of chitinase, that is, the β-GlcNAcase activity in the mycelia decreased with the growth of the fruit-body, whereas that in the culture filtrate increased with the growth. β-GlcNAcase prepared from both mycelia and culture filtrate were partially purified by isoelectrofocusing chromatography and their characteristics were investigated.

Effect of 20-hydroxyecdysone on chitinase and [formula omitted] during the larval-pupal transformation of Manduca sexta (L.)

The relationship between ecdysteroid and chitinolytic enzyme activities was investigated using isolated abdomens from fifth instar larvae of the tobacco hornworm, Manduca sexta (L.). Low levels of 20-hydroxyecdysone (1 μg/g live weight) stimulated exo-splitting β-N- acetylglucosaminidase about four-fold in integument while higher hormonal levels (15 μg/g) which were less effective for β-N- acetylglucosaminidase induced endo-splitting chitinase about 50-fold. Chitinase activity was increased by about ten-fold over the control level in response to injection of 7.5 μg of 20-hydroxyecdysone/g live weight into abdomens, while β-N- acetylglucosaminidase activity was elevated only about 1.5-fold over control. Apparently, ecdysteroid causes synthesis or activation of chitinolytic enzymes in vivo and the major regulatory effect is on the level of the endo-splitting chitinase.

The level of chitinolytic enzymes and ecdysteroids during larval-pupal development in Ephestia cautella and their modifications by a juvenile hormone analogue

The influence of the juvenoid methoprene on ecdysteroid titre and enzymatic activity of N- acetylglucosaminidase and chitinase in Ephestia cautella was examined. Treatment of the larvae in the early fourth larval instar prevents not only the normal rise in ecdysteroid titre but also the increase in activity of both enzymes. Treatment of pupae has no effect on either the ecdysteroid titre or on the enzymatic activities. Larvae treated with methoprene during the last quarter of the larval stage show intermediate values in ecdysteroid titre as well as in chitinolytic enzyme activities. A causal relationship between moulting hormone titre and activity of chitin-degrading enzyme is proposed.