Showing papers on "Chitinase published in 1999"

Chitin and chitinases.

Abstract: Native, industrial and fossil chitins.- Chitin synthesis.- Biochemistry of chitin synthase.- Chitin biosynthesis and structural organizationin vivo.- Chitin synthases in yeast and fungi.- Function of chitin oligosaccharides in plant and animal development.- Molecular and biochemical aspects of chitin synthesis inhibition.- Characteristics of chitin-binding proteins from streptomycetes.- Chitinases.- Biochemistry of chitinases.- The structure and action of chitinases.- Classification of chitinase modules.- Aggressive and defensive roles for chitinases.- Chitinases in biological control.- Host-parasite interactions: elicitation of defense responses in plants with chitosan.- Inhibitors of chitinases.- Mammalian chitinase-like proteins.- Chitinases of human parasites and their implications as antiparasitic targets.- Analytical biochemistry and clinical significance of N-acetyl-ss-D-glucosaminidase and related enzymes.- Exogenous chitosans.- Biochemistry, histology and clinical uses of chitins and chitosans in wound healing.- Veterinary practice with chitin and chitosan.- Immunological aspects of chitin and chitin derivatives administered to animals.- Clinical and biochemical evaluation of chitosan for hypercholesterolemia and overweight control.- Microparticulate drug delivery systems.- Antimicrobial action of exogenous chitosan.

Chitinases from uncultured marine microorganisms.

Abstract: Our understanding of the degradation of organic matter will benefit from a greater appreciation for the genes encoding enzymes involved in the hydrolysis of biopolymers such as chitin, one of the most abundant polymers in nature. To isolate representative and abundant chitinase genes from uncultivated marine bacteria, we constructed libraries of genomic DNA isolated from coastal and estuarine waters. The libraries were screened for genes encoding proteins that hydrolyze a fluorogenic analogue of chitin, 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside (MUF-diNAG). The abundance of clones capable of MUF-diNAG hydrolysis was higher in the library constructed with DNA from the estuary than in that constructed with DNA from coastal waters, although the abundance of positive clones was also dependent on the method used to screen the library. Plaque assays revealed nine MUF-diNAG-positive clones of 75,000 screened for the estuarine sample and two clones of 750,000 for the coastal sample. A microtiter plate assay revealed approximately 1 positive clone for every 500 clones screened in the coastal library. The number of clones detected with the plaque assay was consistent with estimates of the portion of culturable bacteria that degrade chitin. Our results suggest that culture-dependent methods do not greatly underestimate the portion of marine bacterial communities capable of chitin degradation.

Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by constitutive expression of rice chitinase.

Abstract: Rice blast is the most devastating plant disease in Japan. Our goal is to create new rice varieties which show enhanced resistance against blast, regardless of the race of blast. By an Agrobacterium-mediated transformation method, we reintroduced a rice class-I chitinase gene, Cht-2 or Cht-3, under the control of the enhanced CaMV 35S promoter and a hygromycin phosphotransferase gene, as a selection marker into the Japonica rice varieties Nipponbare and Koshihikari, which have retained the best popularity over a long period in Japan. In regenerated plants (R0), the Cht-2 product was found to accumulate intracellularly whereas the Cht-3 product was found to be targeted extracellularly. The transgenic rice plants which constitutively expressed either chitinase gene showed significantly higher resistance against the rice blast pathogen Magnaporthe grisea races 007.0 and 333. Both high-level expression of the chitinase and blast-resistance were stably inherited by the next generation in several lines.

The role of an extracellular chitinase from Trichoderma virens Gv29-8 in the biocontrol of Rhizoctonia solani.

Abstract: The role of extracellular chitinase in the biocontrol activity of Trichoderma virens was examined using genetically manipulated strains of this fungus. The T. virens strains in which the chitinase gene (cht42) was disrupted (KO) or constitutively over-expressed (COE) were constructed through genetic transformation. The resulting transformants were stable and showed patterns similar to the wild-type (WT) strain with respect to growth rate, sporulation, antibiotic production, colonization efficiency on cotton roots and growth/survival in soil. Biocontrol activity of the KO and COE strains were significantly decreased and enhanced, respectively against cotton seedling disease incited by Rhizoctonia solani when compared with the WT strain.

Role of the Trichoderma harzianum endochitinase gene, ech42, in mycoparasitism.

Abstract: The role of the Trichoderma harzianum endochitinase (Ech42) in mycoparasitism was studied by genetically manipulating the gene that encodes Ech42, ech42. We constructed several transgenic T. harzianum strains carrying multiple copies of ech42 and the corresponding gene disruptants. The level of extracellular endochitinase activity when T. harzianum was grown under inducing conditions increased up to 42-fold in multicopy strains as compared with the wild type, whereas gene disruptants exhibited practically no activity. The densities of chitin labeling of Rhizoctonia solani cell walls, after interactions with gene disruptants were not statistically significantly different than the density of chitin labeling after interactions with the wild type. Finally, no major differences in the efficacies of the strains generated as biocontrol agents against R. solani or Sclerotium rolfsii were observed in greenhouse experiments.

The third chitinase gene (chiC) of Serratia marcescens 2170 and the relationship of its product to other bacterial chitinases.

Abstract: The third chitinase gene (chiC) of Serratia marcescens 2170, specifying chitinases C1 and C2, was identified. Chitinase C1 lacks a signal sequence and consists of a catalytic domain belonging to glycoside hydrolase family 18, a fibronectin type III-like domain (Fn3 domain) and a C-terminal chitin-binding domain (ChBD). Chitinase C2 corresponds to the catalytic domain of C1 and is probably generated by proteolytic removal of the Fn3 and ChBDs. The loss of the C-terminal portion reduced the hydrolytic activity towards powdered chitin and regenerated chitin, but not towards colloidal chitin and glycol chitin, illustrating the importance of the ChBD for the efficient hydrolysis of crystalline chitin. Phylogenetic analysis showed that bacterial family 18 chitinases can be clustered in three subfamilies which have diverged at an early stage of bacterial chitinase evolution. Ser. marcescens chitinase C1 is found in one subfamily, whereas chitinases A and B of the same bacterium belong to another subfamily. Chitinase C1 is the only Ser. marcescens chitinase that has an Fn3 domain. The presence of multiple, divergent, chitinases in a single chitinolytic bacterium is perhaps necessary for efficient synergistic degradation of chitin.

Genetic engineering of wheat for increased resistance to powdery mildew disease

Abstract: Fungal wheat (Triticum aestivum) diseases greatly affect crop productivity and require the economically and ecologically undesirable application of fungicides in wheat agriculture. We have generated transgenic wheat plants constitutively expressing an antifungal barley-seed class II chitinase. The transgene was stably expressed and the chitinase properly localized in the apoplast of the transgenic lines. The engineered wheat plants showed increased resistance to infection with the powdery mildew-causing fungus Erysiphe graminis.

A unique chitinase with dual active sites and triple substrate binding sites from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1.

Abstract: We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. The chiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85°C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA.

Family 19 chitinases of Streptomyces species: characterization and distribution.

Abstract: Chitinase C from Streptomyces griseus HUT6037, described in 1997, is the first family 19 chitinase found in an organism other than higher plants. In this study, some properties of chitinase C were compared with those of family 18 bacterial chitinases, and the distribution of family 19 chitinases in Streptomyces species was investigated. The specific hydrolysing activity of chitinase C against soluble and insoluble chitinous substrates was markedly higher than those of bacterial family 18 chitinases. Chitinase C exhibited marked antifungal activity, whereas the other bacterial chitinases examined had no antifungal activity. Chitinase C was insensitive to allosamidin, whereas the family 18 bacterial chitinases were sensitive. Taking advantage of this insensitivity to allosamidin, a search was made for family 19 chitinases in various Streptomyces species. Chitinases insensitive to allosamidin were detected in the culture supernatants of all tested Streptomyces species. Southern hybridization analysis using a labelled DNA fragment corresponding to the catalytic domain of chitinase C strongly suggested that these species have genes similar to the chiC gene of S. griseus HUT6037. DNA fragments corresponding to the major part of the catalytic domains were amplified by PCR. The amplified fragments encoded amino acid sequences very similar to that of the corresponding region of chitinase C. Therefore, it was concluded that Streptomyces species generally possess family 19 chitinases which are very similar to chitinase C. Comparison of their amino acid sequences with those of plant family 19 chitinases revealed that Streptomyces family 19 chitinases are class IV type in terms of the presence and positions of deletions of amino acid sequences which are characteristic of plant class IV chitinases.

Expression of two major chitinase genes of Trichoderma atroviride (T. harzianum P1) is triggered by different regulatory signals.

Abstract: Regulation of the expression of the two major chitinase genes, ech42 (encoding the CHIT42 endochitinase) and nag1 (encoding the CHIT73 N-acetyl-β-d-glucosaminidase), of the chitinolytic system of the mycoparasitic biocontrol fungus Trichoderma atroviride (= Trichoderma harzianum P1) was investigated by using a reporter system based on the Aspergillus niger glucose oxidase. Strains harboring fusions of the ech42 or nag1 5′ upstream noncoding sequences with the A. niger goxA gene displayed a glucose oxidase activity pattern that was consistent under various conditions with expression of the native ech42 and nag1 genes, as assayed by Northern analysis. The expression product of goxA in the mutants was completely secreted into the medium, detectable on Western blots, and quantifiable by enzyme-linked immunosorbent assay. nag1 gene expression was triggered during growth on fungal (Botrytis cinerea) cell walls and on the chitin degradation product N-acetylglucosamine. N-Acetylglucosamine, di-N-acetylchitobiose, or tri-N-acetylchitotriose also induced nag1 gene expression when added to mycelia pregrown on different carbon sources. ech42 expression was also observed during growth on fungal cell walls but, in contrast, was not triggered by addition of chitooligomers to pregrown mycelia. Significant ech42 expression was observed after prolonged carbon starvation, independent of the use of glucose or glycerol as a carbon source, suggesting that relief of carbon catabolite repression was not involved in induction during starvation. In addition, ech42 gene transcription was triggered by physiological stress, such as low temperature, high osmotic pressure, or the addition of ethanol. Four copies of a putative stress response element (CCCCT) were found in the ech42 promoter.

Induction of different pathogenesis-related cDNAs in grapevine infected with powdery mildew and treated with ethephon

Abstract: Uncinula necator, the causal agent of grapevine powdery mildew, was found to elevate the activity of the pathogenesis-related (PR) proteins, chitinase and β-1,3-glucanase in leaves and berries of a number of susceptible grapevine (Vitis vinifera) cultivars. The increase in hydrolytic activity was directly related to the severity of powdery mildew infection on both leaves and berries but no systemic induction was observed in uninfected tissues. A number of cDNA clones encoding various PR proteins, including chitinases (PR-2), β-1,3-glucanases (PR-3) and thaumatin-like (TL) proteins (PR-5), were isolated and their expression in response to powdery mildew infection examined. PR genes encoding extracellular proteins were most strongly induced in infected leaves, including an acidic class III chitinase (VvChi3), a basic class I glucanase (VvGlub) and a thaumatin-like protein (VvTl2). A basic class I chitinase (VvChi1b) was also detected, but only in severely infected leaves. A similar pattern of PR gene induction was observed in mildew-infected preveraison berries, with the exception of VvChi1b, which was not detected. Chitinase and β-1,3-glucanase enzyme activities were also found to be highly induced in leaves and preveraison berries by ethephon treatment. The pattern of chitinase gene induction by ethephon, however, was markedly different for mildew infection, with no apparent induction of VvChi3 but a marked increase in the transcript level of an acidic class IV chitinase, VvChi4. These results demonstrate that the leaves and preveraison berries of grapevine respond to environmental and stress-related stimuli by the expression of specific PR genes but that the induction of these genes during pathogen invasion does not afford complete protection of these tissues against Uncinula necator. The following abbreviations have been used: Tris: 2-amino-2-(hydroxymethyl)-1,3-propanediol; EDTA: ethylenediaminetetra-acetic acid; PVPP: polyvinylpolypyrrolidone. The GenBank accession numbers for VvChi1b and VvGlub are AF053341 and AF053750, respectively.

Increased antifungal activity of Trichoderma harzianum transformants that overexpress a 33-kDa chitinase

Abstract: Transformants of the biocontrol agent Trichoderma harzianum strain CECT 2413 that overexpressed a 33-kDa chitinase (Chit33) were obtained and characterized. Strain CECT 2413 was cotransformed with the amdS gene and its own chit33 gene under the control of the pki constitutive promoter from T. reesei. Southern blotting indicated that the chit33 gene was integrated ectopically, mostly in tandem. Some transformants showed the same restriction pattern, indicating preferable sites of integration. There was no correlation between the number of integrated copies and the level of expression of the chit33 gene in the transformants. When grown in glucose, the extracellular chitinase activity of the transformants was up to 200-fold greater than that of the wild type, whereas in chitin, the activity of both the transformants and the wild type was similar. Under both conditions, the transformants were more effective in inhibiting the growth of Rhizoctonia solani as compared with the wild type. Similar results were obtained when culture supernatants from the transformants and the wild type were tested against R. solani.

Class I chitinases as potential panallergens involved in the latex-fruit syndrome

Abstract: Background: Latex-fruit cross-sensitization has been fully demonstrated. However, the antigens responsible for this "latex-fruit syndrome" have not been identified. We have recently shown that class I chitinases are relevant chestnut and avocado allergens. Objective: We sought to evaluate the in vivo and in vitro reactions of purified chestnut and avocado chitinases in relation to the latex-fruit syndrome. Methods: From a latex-allergic population, eighteen patients allergic to chestnut, avocado, or both were selected. Skin prick tests (SPTs) were performed with crude chestnut and avocado extracts, chitinase-enriched preparations, and purified class I and II chitinases from both fruits. CAP-inhibition assays with the crude extracts and purified proteins were carried out. Immunodetection with sera from patients with latex-fruit allergy and immunoblot inhibition tests with a latex extract were also performed. Eighteen subjects paired with our patients and 15 patients allergic to latex but not food were used as control groups. Results: The chestnut class I chitinase elicited positive SPT responses in 13 of 18 patients with latex-fruit allergy (72%), and the avocado class I chitinase elicited positive responses in 12 of 18 (67%) similarly allergic patients. By contrast, class II enzymes without a hevein-like domain did not show SPT responses in the same patient group. Each isolated class I chitinase reached inhibition values higher than 85% in CAP inhibition assays against the corresponding food extract in solid phase. Immunodetection of the crude extracts and the purified class I chitinases revealed a single 32-kd band for both chestnut and avocado. Preincubation with a natural latex extract fully inhibited the IgE binding to the crude extracts, as well as to the purified chestnut and avocado class I chitinases. Conclusion: Chestnut and avocado class I chitinases with an N-terminal hevein-like domain are major allergens that cross-react with latex. Therefore they are probably the panallergens responsible for the latex-fruit syndrome. (J Allergy Clin Immunol 1999;103:507-13.)

Cross-reactions in the latex-fruit syndrome: A relevant role of chitinases but not of complex asparagine-linked glycans

Abstract: Background: Cross-reactions between latex and plant foods (mainly fruits) have been widely reported. Although the cross-reactive components have not been well identified, class I chitinases seem to be the most credible candidates in chestnut, avocado, and banana. Objective: We sought to evaluate the potential role of chitinases and complex glycans as cross-reactive determinants linked to latex-food allergy. Methods: Extracts from 20 different plant foods and from latex were obtained. These preparations were immunodetected with anticomplex glycans and antichitinase sera raised in rabbits, as well as with sera from patients with latex-fruit allergy and sera from patients allergic to latex without food allergy. Immunoblot inhibition assays were carried out by using a purified class I chitinase from avocado or latex extract as inhibitors. Results: Reactive proteins of approximately 30 to 45 kd (putative class I chitinases) were recognized by both specific polyclonal antibodies to chitinases and sera from patients with latex-fruit allergy in chestnut, cherimoya, passion fruit, kiwi, papaya, mango, tomato, and flour wheat extracts. Prs a 1, the major allergen and class I chitinase from avocado, and the latex extract strongly or fully inhibited IgE binding by these components when tested in immunoblot inhibition assays. Additional bands of 16 to 20 kd, 23 to 28 kd, and 50 to 70 kd were detected by the antichitinase serum but not with the patients' pooled sera. The putative 30- to 45-kd chitinases present in different food extracts did not react with a pool of sera from subjects allergic to latex but not to fruit. Very different immunodetection patterns were produced with the anticomplex glycan serum and the sera from allergic patients. Conclusions: Putative class I chitinases seem to be relevant cross-reactive components in foods associated with the latex-fruit syndrome, but do not play a specific role in allergy to latex but not to fruit. Cross-reactive carbohydrate determinants are not important structures in the context of latex-fruit cross-sensitization. (J Allergy Clin Immunol 1999;104:681-7.)

The chitinase PfCHT1 from the human malaria parasite Plasmodium falciparum lacks proenzyme and chitin-binding domains and displays unique substrate preferences

Abstract: Within hours after the ingestion of a blood meal, the mosquito midgut epithelium synthesizes a chitinous sac, the peritrophic matrix. Plasmodium ookinetes traverse the peritrophic matrix while escaping the mosquito midgut. Chitinases (EC 3.2.1.14) are critical for parasite invasion of the midgut: the presence of the chitinase inhibitor, allosamidin, in an infectious blood meal prevents oocyst development. A chitinase gene, PgCHT1, recently has been identified in the avian malaria parasite P. gallinaceum. We used the sequence of PgCHT1 to identify a P. falciparum chitinase gene, PfCHT1, in the P. falciparum genome database. PfCHT1 differs from PgCHT1 in that the P. falciparum gene lacks proenzyme and chitin-binding domains. PfCHT1 was expressed as an active recombinant enzyme in Escherichia coli. PfCHT1 shares with PgCHT1 a substrate preference unique to Plasmodium chitinases: the enzymes cleave tri- and tetramers of GlcNAc from penta- and hexameric oligomers and are unable to cleave smaller native chitin oligosaccharides. The pH activity profile of PfCHT1 and its IC50 (40 nM) to allosamidin are distinct from endochitinase activities secreted by P. gallinaceum ookinetes. Homology modeling predicts that PgCHT1 has a novel pocket in the catalytic active site that PfCHT1 lacks, which may explain the differential sensitivity of PfCHT1 and PgCHT1 to allosamidin. PfCHT1 may be the ortholog of a second, as yet unidentified, chitinase gene of P. gallinaceum. These results may allow us to develop novel strategies of blocking human malaria transmission based on interfering with P. falciparum chitinase.

Regulation and cloning of microbial chitinase genes.

Abstract: A range of chitinase genes from microorganisms have been cloned and the potential uses of these genetically manipulated organisms are being investigated by various researchers Fungi and yeast are better producers of chitinase than bacteria Since fungi grow at a slower rate, there have been efforts to clone the fungal chitinase genes into fast-growing bacteria This review gives a brief survey of recent progress in the regulation and cloning of microbial chitinase genes Emphasis is placed on the post-translational modification and localization of the recombinant protein in the host Various amino acid domains are present in this protein The mode of catalytic activity of the recombinant protein in comparison to the wild-type protein is discussed in the available literature The different mechanisms involved in the regulation of chitinase genes from various microorganisms is discussed by the researchers The scope of future research and conclusions yet to be obtained in this particular area are also outlined in this review

Regulation of the chitinase gene expression in suspension-cultured rice cells by N-acetylchitooligosaccharides: differences in the signal transduction pathways leading to the activation of elicitor-responsive genes.

Abstract: Expression patterns of chitinase transcripts induced by N-acetylchitooligosaccharide elicitor were analyzed by northern blot hybridization in order to reveal a signal transduction pathway leading to the activation of class I chitinase genes (Cht-1 and Cht-3), which may play an important role in producing N-acetylchitooligosaccharide elicitor. The transcription level of both genes was enhanced in response to N-acetylchitooligosaccharides larger than pentaose at subnanomolar concentrations. These structure and dose dependencies were consistent not only with those for a 75 kDa high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane, but also with other series of cellular responses including phytoalexin production and the expression of elicitor-responsive genes (EL2, EL3). Therefore, the elicitor signal to evoke these cellular responses including the activation of the chitinase genes could be common and transmitted into cells through the 75 kDa protein. However, the signal transduction pathway for the activation of the chitinase gene appeared to diverge from those for the other elicitor-responsive genes shortly after the signal perception. It was shown that the induction of chitinase expression by N-acetylchitooligosaccharide would require protein phosphorylation, but not de novo protein synthesis. The oxidative burst was demonstrated not to be necessary for transcriptional induction of the all four elicitor-responsive genes (Cht, PAL, EL2, EL3) by N-acetylchitooligosaccharide.

Differential Induction of Grapevine Defenses by Two Strains of Botrytis cinerea

Abstract: Even though Botrytis cinerea, the causal agent of gray mold, is a highly variable fungus with strains displaying very different degrees of virulence toward one given host plant species, no study has yet shown any correlation between the lack of aggressiveness of one given strain and its ability to stimulate a defense response from its host. Strains of B. cinerea collected from different host plant species were screened for their pathogenicity on grapevine to select two strains with similar morphological characteristics but different levels of virulence. In grapevine leaves, the less aggressive strain, T4, enhanced the accumulation of many defense products including secondary metabolites and the pathogenesis-related proteins, chitinase and beta-1,3-glucanase. Interestingly, secondary metabolites were formed in cells around a small group of dead cells. When compared with T4, the more aggressive strain, T8, had larger necrotic spots, no secondary metabolite biosynthesis, and accumulations of chitinases and beta-1,3-glucanases that were more delayed, yet only slightly weaker. The culture fluids of both strains mimicked the differential effect of each isolate in stimulating chitinase activity when infiltrated into grapevine leaves.

High-multiplicity of Chitinase genes in Streptomyces coelicolor A3(2)

Abstract: Six different genes for chitinase from ordered cosmids of the chromosome of Streptomyces coelicolor A3(2) were identified by hybridization, using the chitinase genes from other Streptomyces spp. as probes, and cloned. The genes were sequenced and analyzed. The genes, together with an additional chitinase gene obtained from the data bank, can be classified into either family 18 or family 19 of the glycosyl hydrolase classification. The five chitinases that fall into family 18 show diversity in their multiple domain structures as well as in the amino acid sequences of their catalytic domains. The remaining two chitinases are members of family 19 chitinases, since their C-terminus shares more than 70% identity with the catalytic domain of ChiC of Streptomyces griseus, the sole gene for family 19 chitinase so far found in an organism other than higher plants.

Purification and characterization of chitinase from Bacillus circulans No.4.1.

Abstract: Bacillus circulans No.4.1 produced a high level of chitinase when cells were grown in tryptic soy broth supplemented with 0.3% colloidal chitin at 35 degrees C for 5 days. Purification was carried out by protein precipitation with 80% saturation ammonium sulfate, anion-exchange chromatography with DEAE-Sephacel, and gel filtration with Sephadex G-100, sequentially. The purified enzyme could be demonstrated as a single band on SDS-PAGE, estimated to be 45 kDa. This enzyme could hydrolyze colloidal chitin, purified chitin, glycol chitin, carboxymethyl-chitin (CM-chitin), and 4-methylumbelliferyl-beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)(2)]. The optimal conditions for this chitinase were pH 8.0 and 40 degrees C. The isoelectric point of the chitinase was 5.1. The amino acid composition of the purified chitinase was determined. The initial 20 amino acid residues of the N-terminal were found to be alanine (A), proline (P), tryptophan (W), asparagine (N), serine (S), lysine (K), glycine (G), asparagine (N), tyrosine (Y), alanine (A), leucine (L), proline (P), tyrosine (Y), tyrosine (Y), arginine (R), glycine (G), alanine (A), tryptophan (W), alanine (A), and valine (V). Knowledge of these properties of chitinase from B. circulans No. 4.1 should be useful in the development of genetically engineered Bacillus sp. as biopesticides.

Response of the chitinolytic microbial community to chitin amendments of dune soils

Abstract: The dynamics of culturable chitin-degrading microorganisms were studied during a 16-week incubation of chitin-amended coastal dune soils that differed in acidity. Soil samples were incubated at normal (5% w/w) and high (15% w/w) moisture levels. More than half of the added chitin was decomposed within 4 weeks of incubation in most soils. This rapid degradation was most likely due to fast-growing chitinolytic fungi (mainly Mortierella spp. and Fusarium spp.) at both moisture levels, as dense hyphal networks of these fungi were observed during the first 4 weeks of incubation. Chitin N mineralization was inhibited by cycloheximide, and fast-growing fungal isolates were capable of rapid chitin decomposition in sterile sand, further suggesting that these fungi play an important role in initial chitin degradation. The strong increase in fast-growing fungi in chitin-amended dune soils was only detected by direct observation. Plate counts and microscopic quantification of stained hyphae failed to reveal such an increase. During the first part of the incubation, numbers of unicellular chitinolytic bacteria also increased, but their contribution to chitin degradation was indicated to be of minor importance. During prolonged incubation, colony forming units (CFU) of chitinolytic streptomycetes and/or slow-growing fungi increased strongly in several soils, especially at the 5% moisture level. Hence, the general trend observed was a succession from fast-growing fungi and unicellular bacteria to actinomycetes and slow-growing fungi. Yet, the composition of chitinolytic CFU over time differed strongly between chitin-amended dune soils, and also between the two moisture levels. These differences could not be attributed to pH, organic matter or initial microbial composition. The possible consequence of such unpredictable variation in microbial community composition for the use of chitin-amendments as a biocontrol measure is discussed.

Mammalian chitinase-like proteins.

Abstract: Mammals express genes coding for proteins that show significant similarity to chitinases of family 18 glycosyl hydrolases. These chitinase-like proteins have no chitinase activity due to changes in critical residues in the putative active center. One of these is oviductin, a high molecular weight glycoprotein most likely involved in fertilization and protection of the tubal epithelium owing to its mucin character. Another is YKL-40 (HCgp39) produced in association with tissue remodeling. Such proteins could have a general function in morphogenesis.

Comparison of local and systemic induction of acquired disease resistance in cucumber plants treated with benzothiadiazoles or salicylic acid

Abstract: The accumulation of chitinase and its involvement in systemic acquired disease resistance was analyzed using acibenzolar-S-methyl and salicylic acid (SA). Resistance against scab (pathogen: Cladosporium cucumerinum) and the accumulation of chitinase were rapidly induced in cucumber plants after treatment with acibenzolar-S-methyl. In contrast, SA protected the plants from C. cucumerinum and the accumulation of chitinase was induced only on the treated leaves. The accumulation of chitinase in response to inoculation with the pathogen was induced more rapidly in cucumber plants previously treated with acibenzolar-S-methyl than in plants pretreated with SA or water. Thus, it appears that a prospective signal(s), that induces systemic resistance, can be transferred from leaves treated with acibenzolar-S-methyl to the untreated upper and lower leaves where systemic resistance is elicited. In contrast, exogenously applied SA is not likely to function as a mobile, systemic resistance-inducing signal, because SA only induces localized acquired resistance.