Showing papers on "Chitinase published in 1988"

Antifungal Hydrolases in Pea Tissue II. Inhibition of Fungal Growth by Combinations of Chitinase and β-1,3-Glucanase

Abstract: Chitinase and β-1,3-glucanase purified from pea pods acted synergistically in the degradation of fungal cell walls. The antifungal potential of the two enzymes was studied directly by adding protein preparations to paper discs placed on agar plates containing germinated fungal spores. Protein extracts from pea pods infected with Fusarium solani f.sp. phaseoli, which contained high activities of chitinase and β-1,3-glucanase, inhibited growth of 15 out of 18 fungi tested. Protein extracts from uninfected pea pods, which contained low activities of chitinase and β-1,3-glucanase, did not inhibit fungal growth. Purified chitinase and β-1,3-glucanase, tested individually, did not inhibit growth of most of the test fungi. Only Trichoderma viride was inhibited by chitinase alone, and only Fusarium solani f.sp. pisi was inhibited by β-1,3-glucanase alone. However, combinations of purified chitinase and β-1,3-glucanase inhibited all fungi tested as effectively as crude protein extracts containing the same enzyme activities. The pea pathogen, Fusarium solani f.sp. pisi, and the nonpathogen of peas, Fusarium solani f.sp. phaseoli, were similarly strongly inhibited by chitinase and β-1,3-glucanase, indicating that the differential pathogenicity of the two fungi is not due to differential sensitivity to the pea enzymes. Inhibition of fungal growth was caused by the lysis of the hyphal tips.

Plant and bacterial chitinases differ in antifungal activity

Abstract: SUMMARY: Chitinases were isolated from the grains of wheat, barley and maize, and compared with those obtained from Serratia marcescens, Streptomyces griseus and Pseudomonas stutzeri for antifungal activity and enzyme specificity. The six enzymes were tested for antifungal activity using an assay based upon inhibition of hyphal extension of the fungi Trichoderma reesei and Phycomyces blakesleeanus. Antifungal activity was observed with as little as 1 μg of each of the grain chitinases, whereas none of the bacterial chitinases had any effect on hyphal extension, even at 50 μg chitinase per assay. This difference in antifungal activity correlated with the different mechanisms of action of the two classes of enzymes. In common with other plant chitinases, the grain chitinases functioned as endochitinases and contained lysozyme activity. In contrast, the bacterial enzymes were exochitinases and hydrolysed the chromogenic trisaccharide analogue p-nitrophenyl-β-D-N, N'-diacetylchitobiose, which proved to be an excellent substrate for assaying bacterial chitinases. These experiments strengthen the hypothesis that plant chitinases function to protect the host against fungal infections.

Several “pathogenesis-related” proteins in potato are 1,3-β-glucanases and chitinases

Abstract: Chitinase {poly[1,4-(N-acetyl-β-D-glucosaminide)]glycanohydrolase, EC 3.2.1.14} and 1,3-β-glucanase (1,3-β-D-glucan 3-glucanohydrolase, EC 3.2.1.6) activities increased rapidly in potato (Solanum tuberosum) leaves inoculated with the pathogenic fungus Phytophthora infestans or treated with fungal elicitor. The enzyme activities were resolved into a total of two distinct 1,3-β-glucanases and six proteins with chitinase activity. By several criteria, all of these proteins are classified as “pathogenesis-related” proteins whose biochemical functions have so far been unknown. Some of them constitute a major portion of the proteins accumulating in the intercellular space of infected potato leaves and are assumed to play an important role in pathogen defense.

Antifungal Hydrolases in Pea Tissue: I. Purification and Characterization of Two Chitinases and Two β-1,3-Glucanases Differentially Regulated during Development and in Response to Fungal Infection

Abstract: Chitinase and β-1,-3-glucanase activities increased coordinately in pea (Pisum sativum L. cv “Dot”) pods during development and maturation and when immature pea pods were inoculated with compatible or incompatible strains of Fusarium solani or wounded or treated with chitosan or ethylene. Up to five major soluble, basic proteins accumulated in stressed immature pods and in maturing untreated pods. After separation of these proteins by chromatofocusing, an enzymic function could be assigned to four of them: two were chitinases and two were β-1,3-glucanases. The different molecular forms of chitinase and β-1,3-glucanase were differentially regulated. Chitinase Ch1 (mol wt 33,100) and β-1,3-glucanase G2 (mol wt 34,300) were strongly induced in immature tissue in response to the various stresses, while chitinase Ch2 (mol wt 36,200) and β-1,3-glucanase G1 (mol wt 33,500) accumulated during the course of maturation. With a simple, three-step procedure, both chitinases and both β-1,3-glucanases were purified to homogeneity from the same extract. The two chitinases were endochitinases. They differed in their pH optimum, in specific activity, in the pattern of products formed from [3H]chitin, as well as in their relative lysozyme activity. Similarly, the two β-1,3-glucanases were endoglucanases that showed differences in their pH optimum, specific activity, and pattern of products released from laminarin.

Colorimetric assay for chitinase

Abstract: Publisher Summary This chapter describes the colorimetric assay for chitinase, which is applicable to the various types of chitinase present in microorganisms, animals, and plants The chapter evaluates it with particular reference to plant chitinases, because they may function as a defense against chitin-containing pathogens The most widely used colorimetric assay for plant chitinases has been an exochitinase assay, based on the determination of monomeric N-acetylglucosamine (GlcNAc) released from colloidal chitin However, plant chitinases generally are endochitinases and produce chitooligosaccharides as principal products Therefore, measurements of plant chitinases with the exochitinase assay should be viewed with caution For accurate determination, it is essential to measure the chitooligosaccharides produced in the assay This can be accomplished by the enzymatic hydrolysis of the reaction products to monomeric GlcNAc prior to the colorimetric measurement The chapter compares the assay with other chitinase assays

Preparation of crustacean chitin

Abstract: Publisher Summary This chapter describes the isolation of chitin from crustacean cuticles (shells). To isolate chitin from crustacean shells, the following three steps are required: (1) demineralization with dilute hydrochloric acid or with ethylenediaminetetraacetic acid (EDTA), (2) deproteinization with aqueous sodium hydroxide or by use of the proteolytic activity of a bacterium, and (3) elimination of lipids with organic solvent(s). The chapter tabulates composition and molecular weight of various chitin preparations from abdominal shell of Penaeus japonicus (kuruma prawn). The chapter also describes the preparation of colloidal chitin. The procedure consists of the dissolution of chitin into and the reprecipitation from concentrated hydrochloric acid and the dispersion of the reprecipitated chitin into water. Colloidal chitin serves as a substrate of chitinase and a carbon and nitrogen source of chitinolytic microorganisms.

Chitinase cDNA Cloning and mRNA Induction by Fungal Elicitor, Wounding, and Infection

Abstract: Chitinase, which catalyzes the hydrolysis of β-1,4 N-acetylglucosamine linkages of the fungal cell wall polymer chitin, is a component of the inducible defenses of plants. We show that chitinase synthesis is stimulated in bean (Phaseolus vulgaris L.) cell suspension cultures treated with fungal cell wall elicitors and in hypocotyls in response to infection with the fungus Colletotrichum lindemuthianum. Chitinase cDNA clones were isolated by antibody screening of a λgt11 cDNA library containing sequences complementary to poly A+ RNA from elicited cells. The identity of these clones was confirmed by nucleotide sequence analysis and comparison of the deduced amino acid sequence with that determined for the amino-terminal sequence of bean chitinase. Elicitor causes a very rapid activation of chitinase transcription with a 10-fold stimulation after 5 minutes and 30-fold increase within 20 minutes. This leads to a marked, transient accumulation of chitinase transcripts with maximum levels 2 hours after elicitor treatment, concomitant with the phase of rapid enzyme synthesis. Chitinase transcripts also markedly accumulate in wounded and infected hypocotyls. Chitinase cDNA sequences hybridize to several genomic fragments suggesting there are several chitinase genes in the bean genome.

Chitinases and β-1,3-Glucanases in the Apoplastic Compartment of Oat Leaves (Avena sativa L.)

Abstract: To isolate chitinases and β-1,3-glucanases from the intercellular space of oats (Avena sativa L.), primary leaves were infiltrated with buffer and subjected to gentle centrifugation to obtain intercellular washing fluid (IWF). Approximately 5% of the chitinase and 10% of the β-1,3-glucanase activity of the whole leaf were released. Only small amounts (0.01-0.03%) of the intracellular marker malate-dehydrogenase were released into the IWF during infiltration. Activities of chitinase and β-1,3-glucanase in the IWF and in the leaf extract were compared by different chromatographic methods. On Sephadex G-75, chitinase appeared as a single peak (Mr 29.8 kD) both in IWF and homogenate. β-1,3-Glucanase, however, showed two peaks in the IWF (Mr 52 and 31.3 kD), whereas the elution pattern of the homogenate showed only one major peak at 22 kD. Chromatofocusing indicated that the IWF contained four chitinases and five β-1,3-glucanases. The elution pattern of the homogenate and IWF were similar with regard to the elution pH, but the peak intensities were distinctly different. Our results demonstrate that extracellular β-1,3-glucanases are different from those located intracellularly. Extracellular and intracellular chitinases do not differ in molecular properties, except for one isozyme which seems to be confined to the extracellular space. We suggest that both enzymes might play a special role in pathogenesis during fungal infection.

Evidence for a glycosidic linkage between chitin and glucan in the cell wall of Candida albicans.

Abstract: Summary: The alkali-insoluble glucan was isolated from regenerating spheroplasts and intact cells of Candida albicans. Sequential enzymic hydrolysis of this fraction by Zymolyase 100T and purified chitinase and subsequent gel filtration produced a fraction which was enriched in glycosaminoglycans. This fraction was analysed by partial acid hydrolysis, TLC and GLC-MS. The GLC-MS peaks identified included 2,3,4,6-tetra-O-methylglucitol acetate and 2,3,4-tri-O-methylglucitol acetate of β-1,6-glucan and the 3,6-di-O-methyl-2-N-methylglucosaminitol acetate of chitin. In addition, 3-O-methyl-2-N-methylglucosaminitol acetate was identified, which indicated a branch point in chitin. These data provide evidence for a covalent linkage between chitin and β-(1,6)-glucan through a glycosidic linkage at position 6 of N-acetylglucosamine and position 1 of the glucose in the glucan.

Co-ordinated regulation of chitinase and β-1,3-glucanase in bean leaves

Abstract: Ethylene induced chitinase (EC 3.2.1.14) and β-1,3-glucanase (EC 3.2.1.29) to a similar extent in primary leaves of bean seedlings (Phaseolus vulgaris cv. Saxa). Both enzymes were purified from ethylene-treated leaves, and monospecific antibodies were raised aginst them. Ethylene treatments strongly increased the amount of immunore-active chitinase and β-1,3-glucanase. Ethylene enhanced synthesis of chitinase in vivo, as tested by immunoprecipitation after pulse-labelling with [35S]methionine. RNA was isolated from bean leaves and translated in a rabbit reticulocyte lysate system in vitro. The chitinase and the β-1,3-glucanase antiserum each precipitated a single polypeptide from the translation products. The precipitated polypeptides were 1500 and 4000 daltons larger, respectively, than native chitinase and native β-1,3-glucanase, indicating that the two enzymes were synthesized as precursors in vitro. The translatable mRNAs for both enzymes increased at least tenfold within 2 h in response to a treatment with ethylene. When ethylene was withdrawn after 8 h of incubation, the translatable mRNAs for both enzymes decreased somewhat more slowly, reaching the basal level about 25 h later. In all cases, there was a close correlation between the levels of translatable mRNA for chitinase and β-1,3-glucanase. A putative β-1,3-glucanase cDNA clone, pCH16, was isolated by hybrid-selected translation. The amount of β-1,3-glucanase mRNA, as measured by RNA blot analysis using pCH16 as a probe, increased rapidly in response to ethylene and decreased again after withdrawal of ethylene, indicating that the amount of hybridizable RNA and of translatable mRNA for β-1,3-glucanase were correlated. In conclusion, the results indicate that chitinase and β-1,3-glucanase are regulated co-ordinately at the level of mRNA.

Genetic analysis of extracellular proteins of Serratia marcescens.

Abstract: Serratia marcescens, a gram-negative enteric bacterium, is capable of secreting a number of proteins extracellularly. The types of activity found in the growth media include proteases, chitinases, a nuclease, and a lipase. Genetic studies have been undertaken to investigate the mechanisms used for the extracellular secretion of these exoproteins by S. marcescens. Many independent mutations affecting the extracellular enzymes were isolated after chemical and transposon mutagenesis. Using indicator media, we have identified loci involved in the production or excretion of extracellular protease, nuclease, or chitinase by S. marcescens. None of the mutations represented general extracellular-excretion mutants; in no case was the production or excretion of multiple exoproteins affected. A variety of loci were identified, including regulatory mutations affecting nuclease and chitinase expression. A number of phenotypically different protease mutants arose. Some of them may represent different gene products required for the production and excretion of the major metalloprotease, a process more complex than that for the other S. marcescens exoproteins characterized to date.

Chitinase and β-N-acetylhexosaminidase from Pycnoporus cinnabarinus

Abstract: Publisher Summary Pycnoporus cinnabarinus , a strain of the chitinase-producing fungi belonging to the basidiomycetes, has produced the chitinolytic enzymes consisting of chitinase and β-N-acetylhexosaminidase. This chapter describes the procedures for the purification of both enzymes. p-Nitrophenyl-β-N-acetylglucosaminide and β-N-acetylgalactosaminide are used as substrates for purification of β-N-acetylhexosaminidase. The assay for chitinase is based on the estimation of reducing sugars produced in the hydrolysis of colloidal chitin according to a modification of Schales' procedure, with N-acetylglucosamine as a reference compound. The enzyme is purified about 15-fold, starting from the precipitate with ammonium sulfate, and thus purified about 45- to 60-fold over the culture filtrate. The chitinase hydrolyzes colloidal chitin and glycol chitin rapidly and powdered chitin very slowly. Acting upon colloidal chitin, and enzyme produces N,N-diacetylchitobiose as the main product accompanied by a slight formation of N-acetylglucosamine.

Pectic polysaccharides elicit chitinase accumulation in tobacco

Abstract: Upon infection of leaves of tobacco (Nicotiana tabacum L. ev. Havana) with Erwinia carotovora (Jones) Holl, strain 3912, a phytopathogenic bacterium that secretes pectinolytic enzymes, chitinase (EC 3.2.1.14) levels increased 12-fold within 48 h. Heat-killed E. carotovara cells did not induce this response. In young excised tobacco plants supplied with pectic polysaccharides, chitinase activity increased to about the same level as in leaves infected with E. carotovora. The amount of pectic polysaccharides required for half-maximal induction was about 160 μg (g fresh weight)−1. Using in vivo labeling of plants with [35S]-cysteine, it could be demonstrated that elicitormediated chitinase induction is due to enhanced de novo synthesis of the enzyme.

Extracellular enzyme production and cell wall degradation by freshwater lignicolous fungi

Abstract: Ten Ascomycetes, seven Fungi Imperfecti and one Oomycete known to occur on submerged wood were tested for their ability to produce amylase, xylanase, cellulases, lipase, polygalacturonase, pectin lyase, protease, chitinase, and polyphenol oxidases. Species were also tested for their ability to degrade lignosols, with and without wood sugars, and indulin, and to form soft-rot cavities on balsa, green ash, and cottonwood. With the exception of Pythium sp., species were generalists with respect to hydrolytic enzymes and could degrade a wide range of substrates. All species produced weak or negative reactions on media containing chitin and lignin derivatives. Although Nectria haematococca, N. lucidum and Heliscus lugdunensis produced strong to moderate positive reactions on xylan, carboxymethylcellulose and Walseth cellulose, they did not form soft-rot cavities. All other species except Pythium sp. produced typical soft-rot cavities.

Water-soluble glycol chitin and carboxymethylchitin

Abstract: Publisher Summary This chapter describes the procedure for preparation of glycol chitin and carboxymethylchitin. Chitin is treated with aqueous sodium hydroxide solution to give alkali chitin (sodium alkoxides of chitin). The alkali chitin is allowed to react with ethylene chlorohydrin to give glycol [O-(2-hydroxyethyl)] chitin, and with chloroacetic acid to give carboxymethyl [O-carboxymethyl(CM)]chitin. Glycol chitin and CM-chitin are soluble in water and hydrolyzed by chitinase and lysozyme.

Assay for chitinase using tritiated chitin

Abstract: Publisher Summary Chitinase activity has been assayed by a variety of procedures, including the monitoring of changes in the molecular size of substrate by viscosity measurements and the determination of chitooligosaccharides or N-acetylglucosamine liberated in the reaction. This chapter discusses the assay method for chitinase, based on the formation of soluble oligosaccharides from tritium-labeled chitin. The water-soluble oligosaccharides formed are separated from the insoluble chitin by filtration and their radioactivity is determined. This method is by far the most sensitive, because of the possibility of using substrate of very high specific activity. It is suitable for both endo- and exochitinases, it obviates the need for an auxiliary β-N-acetylhexosaminidase, and is extremely simple to carry out.

Glucan–glucosaminoglycan linkages in fungal walls

Abstract: SUMMARY Chemical depolymerization of glucosaminoglycans with nitrous acid solubilized alkali-insoluble glucan from the walls of the basidiomycetes Schizophyllum commune and Agaricus bisporus but not from the walls of the ascomycetes Aspergillus nidulans and Neurospora crassa. In all four fungi depolymerization of glucosaminoglycans with chitinase was effective at converting a considerable portion of the alkali-insoluble glucan into a water/alkali-soluble form. This indicates chemical linkage between glucosaminoglycan (chitin) and s-glucan in these fungi.