Showing papers on "Chitin published in 1988"
TL;DR: With a simple, three-step procedure, both chitinases and both beta-1,3-glucanases were purified to homogeneity from the same extract and showed differences in their pH optimum, specific activity, and pattern of products released from laminarin.
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.
306 citations
TL;DR: This chapter describes the isolation of chitin from crustacean cuticles (shells) and tabulates composition and molecular weight of various chit in preparations from abdominal shell of Penaeus japonicus.
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.
194 citations
TL;DR: The mechanism of in vivo synthesis of chitin has been clarified by cloning the structural gene for the newly found chitIn synthase 2, a relatively minor activity in yeast, establishing that the gene product is essential for both septum formation and cell division.
Abstract: Previous work led to the puzzling conclusion that chitin synthase 1, the major chitin synthase activity in Saccharomyces cerevisiae, is not required for synthesis of the chitinous primary septum. The mechanism of in vivo synthesis of chitin has now been clarified by cloning the structural gene for the newly found chitin synthase 2, a relatively minor activity in yeast. Disruption of the chitin synthase 2 gene results in the loss of well-defined septa and in growth arrest, establishing that the gene product is essential for both septum formation and cell division.
187 citations
TL;DR: Cell wall analysis evidenced a five- to ninefold reduction in the amount of chitin in mutants compared with that in the wild-type strain; however, the percentage of beta-glucan that remained insoluble after alkali extraction was considerably reduced in mutant cells.
Abstract: Calcofluor is a fluorochrome that exhibits antifungal activity and a high affinity for yeast cell wall chitin. We isolated Saccharomyces cerevisiae mutants resistant to Calcofluor. The resistance segregated in a Mendelian fashion and behaved as a recessive character in all the mutants analyzed. Five loci were defined by complementation analysis. The abnormally thick septa between mother and daughter cells caused by Calcofluor in wild-type cells were absent in the mutants. The Calcofluor-binding capacity, observed by fluorescence microscopy, in a S. cerevisiae wild-type cells during alpha-factor treatment was also absent in some mutants and reduced in others. Staining of cell walls with wheat germ agglutinin-fluorescein complex indicated that the chitin uniformly distributed over the whole cell wall in vegetative or in alpha-factor-treated cells was almost absent in three of the mutants and reduced in the two others. Cell wall analysis evidenced a five- to ninefold reduction in the amount of chitin in mutants compared with that in the wild-type strain. The total amounts of cell wall mannan and beta-glucan in wild-type and mutant strains were similar; however, the percentage of beta-glucan that remained insoluble after alkali extraction was considerably reduced in mutant cells. The susceptibilities of the mutants and the wild-type strains to a cell wall enzymic lytic complex were rather similar. The in vitro levels of chitin synthase 2 detected in all mutants were similar to that in the wild type. The significance of these results is discussed in connection with the mechanism of chitin synthesis and cell wall morphogenesis in S. cerevisiae.
163 citations
TL;DR: Evidence is provided for a covalent linkage between chitin and beta-(1,6)-glucan through a glycosidic linkage at position 6 of N-acetylglucosamine and position 1 of the glucose in the glucan.
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.
113 citations
TL;DR: An improved method for fractionating cell-free extracts of Saccharomyces cerevisiae to separate its membranous components by a combination of isopycnic and velocity sedimentations is described, which regards these vesicles as precursors of the final active form of chitin synthetase whose location in the cell has yet to be unequivocally determined.
Abstract: We describe an improved method for fractionating cell-free extracts of Saccharomyces cerevisiae to separate its membranous components by a combination of isopycnic and velocity sedimentations. These procedures were used to examine the subcellular distribution of chitin synthetase (chitin-UDP acetylglucosaminyltransferase; EC 2.4.1.16) in homogenates from exponentially growing walled cells of a wild-type strain of yeast. Chitin synthetase (Chs1) activity was mainly found in two distinct vesicle populations of nearly equal abundance but with markedly different buoyant densities and particle diameters. One population contained 45-65% of the total chitin synthetase and was identified as chitosomes because of microvesicular size (median diameter = 61 nm) and characteristic low buoyant density (1.15 g/ml); it also lacked 1,3-beta-glucan synthetase activity. The second population (35-55%) was identified as plasma membrane because of its high buoyant density (1.22 g/ml), large vesicle size (median diameter = 252 nm), and presence of vanadate-sensitive ATPase. This fraction cosedimented with the main peak of 1,3-beta-glucan synthetase. A third, minor population of chitin synthetase particles was also detected. Essentially all of the chitin synthetase in the two vesicle populations was zymogenic; therefore, we regard these vesicles as precursors of the final active form of chitin synthetase whose location in the cell has yet to be unequivocally determined.
77 citations
TL;DR: The enzyme showed an endo-splitting type of activity, and the end product of chitosan degradation contained a mixture of the dimer and trimer of glucosamine, the smallest of the substrates was a tetramer of glucOSamine.
Abstract: A chitosanase was concentrated from the culture broth of Bacillus circulans MH-K1 and was purified to homogeneity by CM-cellulose and gel permeation chromatography. The enzyme has a molecular weight of about 30, 000, its Km is 0.63mg chitosan/ml and its pI is 9.2. The maximum velocity of chitosan degradation by the enzyme was obtained at 50°C when pH was maintained at 6.5. The enzyme was stable within the range of 0-40°C and pH 4.0-9.0. p-Chloromercuribenzoate and the metal ions of Cu2+ Hg2+, Ni2+, and Zn2+ inhibited the enzyme activity.The enzyme degraded chitosan, glycolchitosan and CM-chitosan, but β-1, 4-glucans such as chitin or its derivatives and CM-cellulose were not susceptible to the enzyme.The degree of deacetylation of chitosan significantly affected its susceptibility to the enzyme action. The most susceptible substrate was 80% deacetylated chitosan, and the substrates with less than 40% deacetylation were not affected by the enzyme. It is suggested that the presence of N-acetylglucosamine residues in the molecule of chitosan play an important role in the recognition of the substrate by the enzyme. The enzyme showed an endo-splitting type of activity, and the end product of chitosan degradation contained a mixture of the dimer and trimer of glucosamine. The smallest of the substrates was a tetramer of glucosamine.
75 citations
TL;DR: This chapter describes the procedures for the purification of β-N-acetylhexosaminidase and chitinolytic enzymes of Pycnoporus cinnabarinus, a strain of the chitinase-producing fungi belonging to the basidiomycetes.
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.
69 citations
TL;DR: The growths of Saccharomyces cerevisiae wild-type strain and another strain containing a disrupted structural gene for chitin synthase (chs1::URA3) were affected by Calcofluor, and basal levels of Chs1 and Chs2 increased nine- and fourfold, respectively, through a mechanism dependent on protein synthesis, since the effect was abolished by cycloheximide.
Abstract: The growths of Saccharomyces cerevisiae wild-type strain and another strain containing a disrupted structural gene for chitin synthase (chs1::URA3), defective in chitin synthase 1 (Chs1) but showing a new chitin synthase activity (Chs2), were affected by Calcofluor. To be effective, the interaction of Calcofluor with growing cells had to occur at around pH 6. Treatment of growing cells from these strains with the fluorochrome led to an increase in the total levels of Chs1 and Chs2 activities measured on permeabilized cells. During treatment, basal levels (activities expressed in the absence of exogenous proteolytic activation) of Chs1 and Chs2 increased nine- and fourfold, respectively, through a mechanism dependent on protein synthesis, since the effect was abolished by cycloheximide. During alpha-factor treatment, both Chs1 and Chs2 levels increased; however, as opposed to what occurred during the mitotic cell cycle, there was no further increase in Chs1 or Chs2 activities by Calcofluor treatment.
67 citations
TL;DR: Greffage du N-carboxyanhydride du glutamate de γ-methyle sur la chitine dans le melange eau/acetate d'ethyle
Abstract: Greffage du N-carboxyanhydride du glutamate de γ-methyle sur la chitine dans le melange eau/acetate d'ethyle
63 citations
TL;DR: Both chitin and chitosan are copolymers of N-acetylglucosamine and glucosamine, and it has been suggested that for every missing acetyl group there are two water molecules.
Abstract: Publisher Summary Both chitin and chitosan are copolymers of N-acetylglucosamine and glucosamine. Chitin is usually prepared from crustacean waste. During the processing, there will be some deacetylation and some chain scission. The properties of the chitin obtained will depend on the process used, the source of the raw chitinous material, and its treatment before processing. As many studies use chitin/chitosan from a single source or prepared by a single method, the calibration may not apply to other samples. In any attempt to analyze chitin, it must be noted that the chitin may contain unremoved protein, carbohydrates, and salts. Chitosan will also probably contain salts, but it can be purified by dissolving in aqueous acetic acid, filtering, precipitating with sodium hydroxide, and washing with methanol. Chitin and chitosan also bond water tenaciously and it has been suggested that for every missing acetyl group there are two water molecules.
TL;DR: 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, and to form soft-rot cavities on balsa, green ash, and cottonwood.
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.
TL;DR: It appears possible that in addition to the formation of chitosan as a structural element of the cell wall, the secreted enzyme may play a role in the production of shorter chain chitan fragments, which might represent a readily accessible substrate for a secreted chitin deacetylase.
Abstract: Publisher Summary Chitin, a β-1,4-1inked homopolymer of N-acetyl-D-glucosamine, is accompanied in the walls of various fungi by chitosan, a polymer similar to chitin by lacking the N-acetyl groups. Chitosan is most likely synthesized by partial deacetylation of the nascent chain of chitin once the latter is formed by chitin synthase from UDP-GlcNAc. After crystallization the microfibrillar chitin appears to be a relatively poor substrate for the respective enzyme chitin deacetylase. This mode of biosynthesis suggests that the chitin/chitosan complex occurring in fungal cell walls is not a mixture of the two pure polymers but is represented by chitin chains that are deacetylated to a variable degree. The question arises, why in plant pathogens of the genus Colletotrichum can high activities of chitin deacetylase be found in the culture filtrate? It appears possible that in addition to the formation of chitosan as a structural element of the cell wall, the secreted enzyme may play a role in the production of shorter chain chitosan fragments. The chitin deacetylases from M. rouxii and from C lindemuthianum are highly active on oligomers derived from chitin. Similar fragments arise by the action of endochitinases of plant or fungal origin and might represent a readily accessible substrate for a secreted chitin deacetylase.
TL;DR: Hydrogen isotopic analyses of beetle chitin demonstrate the potential for development of a palaeoclimatic tool that can be used to complement current palaeoentomological methods, and demonstrate small interspecific and intraspecific variation in beetles of different habit.
TL;DR: The cell ultrastructure of the VAM fungus Gigaspora margarita was examined, and a decrease of chitin was observed which was correlated to the structural simplification of the fungus wall throughout its life‐cycle, suggesting an involvement of chite in specific biological functions such as mechanical resistance and plasticity.
TL;DR: Findings suggest that the chitin soluble extract (CSE) inhibited in vitro adhesion of C. albicans to human epithelial vaginal cells (VEC), and blocked in vivo attachment to murine vaginal mucosa, thereby preventing candidal infection in these animals.
Abstract: In this study, cell walls from Candida albicans were separated and chitin was isolated from these cell walls. A chitin soluble extract (CSE) prepared from the chitin inhibited in vitro adhesion of C. albicans to human epithelial vaginal cells (VEC), and blocked in vivo attachment to murine vaginal mucosa, thereby preventing candidal infection in these animals. These findings suggest that the CSE acts as an adhesin-like substance.
TL;DR: This chapter describes the procedure for preparation of glycol chitin and carboxymethylchitin, which are soluble in water and hydrolyzed by chit inase and lysozyme.
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.
TL;DR: A survey of major polysaccharides occurring in cell walls of Ophiostoma ulmi and Verticillium alboatrum was performed by means of gold-complexed lectins and glycosidic enzymes, finding some differences in wall carbohydrate composition and distribution.
Abstract: A survey of major polysaccharides occurring in cell walls of Ophiostoma ulmi and Verticillium alboatrum was performed by means of gold-complexed lectins and glycosidic enzymes. Some differences in wall carbohydrate composition and distribution were noted in these fungi. Both chitin and cellulose were found to occur in the walls of 0. ulmi whereas only chitin was present in those of V. albo-atrum. Polygalacturonic acids were identified in both fungi but a greater amount of galactose and N-acetyl-Dgalactosamine residues occurred in the walls of V. albo-atrum. In contrast, a-mannosyl and/or a-glucosyl groups were detected only in 0. ulmi cell walls. /-glucosides and sialic acid were absent in the walls of both fungi.
TL;DR: This chapter describes the dissolution and purification of chitin isolates via the newer anhydrous media through a number of solvent systems that permit filtration, extrusion, and coagulation.
Abstract: Publisher Summary This chapter describes the dissolution and purification of chitin isolates via the newer anhydrous media. There is no clear line of demarcation between chitin isolation and its purification, but the isolation method affects chitin quality and in part determines any additional dissolution and other purification steps required. Many investigators have sought to dissolve and spin chitin to obtain rayon like fibers for comparison with those from cellulose. Hence, a number of solvent systems have been elaborated that permit filtration, extrusion, and coagulation. For many chitin derivatives, such as deacetylation to chitosan or hydrolysis to glucosamine, substantial removal of protein and shell inorganics is usually adequate. However, the current trend toward biomedical applications for chitin as well as the interest in chitin in enzymology has focused still further attention on chitin purification methods.
TL;DR: Chitinolytic bacteria were enumerated and isolated from marine waters and sediments along the highly productive Antarctic Peninsula and low mineralization rates suggest that chitin may be accumulating in Antarctic marine sediments, though animals may also play an important role in Chitin degradation.
Abstract: Chitinolytic bacteria were enumerated and isolated from marine waters and sediments along the highly productive Antarctic Peninsula. Chitinolytic bacteria were found in low concentrations (approximately 1 cell per ml) in the water column and at much higher levels in marine surface sediments (104–105 per g). The predominant chitinolytic bacteria isolated from the water column were identified as psychrophilic Vibrio spp. Rates of chitin mineralization were measured by collection of 14CO2 respired from 14C-labeled chitin synthesized from chitosan and [1-14C]acetic anhydride. Chitin mineralization rates were extremely low in the marine waters analyzed (0.00085–0.0019% of the added label respired in 48 h) and appreciably higher in the marine sediments (0.0039–0.01% per 48 h), suggesting that the sediments are much more important in chitin degradation. Such low mineralization rates suggest that chitin may be accumulating in Antarctic marine sediments, though animals may also play an important role in chitin degradation.
TL;DR: The assay method, based on the formation of soluble oligosaccharides from tritium-labeled chitin, is discussed, which is suitable for both endo- and exochitinases, it obviates the need for an auxiliary β-N-acetylhexosaminidase, and is extremely simple to carry out.
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.
TL;DR: The results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae.
TL;DR: An increasing chitinolytic activity was observed with the incubation time in all the conditions, and synthesis of a β-N-acetylgucosaminidase and endochitinase was detected and the possible induction of these enzymes during A. nidulans autolysis is established.
Abstract: Chitinolytic activity in filtrates of Aspergillus nidulans cultures was studied at the start of the autolysis (maximum dry weight of mycelium) and during autolysis in 24 different media. During the growth the chitinolytic activity was induced only by the presence of ascorbic acid or colloidal chitin in the medium. During autolysis an increasing chitinolytic activity was observed with the incubation time in all the conditions, and synthesis of a β-N-acetylgucosaminidase and endochitinase was detected. The possible induction of these enzymes during A. nidulans autolysis is established.
TL;DR: Cell cultures of non-biting midges and Drosophila melanogaster incorporate glucosamine (GlcN) into chitin and GlcN and N -acetyl-glucosamine(GlcNAc) give higher incorporation rates compared to glucose and mannose.
TL;DR: The results may suggest that strain E‐383a produces an exo‐type chitinase which successively hydrolyses the glycosidic linkages of chitIn into biose units, which may provide a new, selective method for the production of this substance.
Abstract: Vibrio anguillarum strain E-383a, isolated from sea water, accumulated a considerable amount of N,N‘-diacetylchitobiose when it was cultivated in a medium containing colloidal chitin. The maximum conversion of chitin to chitobiose was found to be 40·3%. The rate of chitobiose accumulation was accelerated after the cessation of bacterial growth. Small amounts of N-acetylglucosamine and N,N’,N-triacetylchitotriose were also accumulated but no other saccharides were detected. These results may suggest that strain E-383a produces an exo-type chitinase which successively hydrolyses the glycosidic linkages of chitin into biose units. The exclusive accumulation of chitobiose by the bacterial cells may provide a new, selective method for the production of this substance.
TL;DR: This chapter describes the assay method and purification procedure of chitinase from Serratia marcescens and its properties, which include two closely moving bands, both endowed with chit inase activity.
Abstract: Publisher Summary This chapter describes the assay method and purification procedure of chitinase from Serratia marcescens. The chitinase is assayed by the liberation of tritiated oligosaccharides from [acetyl- 3 H]chitin, with phosphate buffer at pH 6.3, at a final concentration of 0.05 M in the reaction mixture. A unit of chitinase is that amount of enzyme that catalyzes the release of 1 μmol of soluble product in 1 min at 30°. Chitinase is purified by the procedure introduced by Jeuniaux that consists in adsorbing specifically the enzyme on chitin. The chitin-chitinase complex is then incubated at 30° until chitin is digested and the purified enzyme is released into solution. The chapter also describes the properties of chitinase. Upon electrophoresis on nondenaturing polyacrylamide gels the purified preparation yields two closely moving bands, both endowed with chitinase activity. After electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, two major bands are also observed, with apparent molecular weights of 58,000 and 52,500. Minor bands at 40,400 and 21,500 are also detected.
TL;DR: This chapter describes the procedures for the viscosimetric assay of chitinase activity in which water-soluble derivatives of Chitin are used as substrates.
Abstract: Publisher Summary This chapter describes the procedures for the viscosimetric assay of chitinase activity in which water-soluble derivatives of chitin are used as substrates. The viscosimetric assay for chitinase is a more sensitive and effective procedure to detect a flight activity. However, this assay procedure is somewhat troublesome and too time consuming to determine the chitinase activity of numerous samples. The viscosity of a glycol chitin solution varies depending on the degree of polymerization. Some differences have been observed among the values of chitinase activity determined by procedure using different batches of substrates. Therefore, the viscosimetric assay of chitinase activity should be carried out using the same batch of glycol chitin in the same experiment. This assay has been applied to the determination of chitinase activity in the purification of chitinases from Aspergillus niger and Vibrio sp., and those from goat serum and bovine serum.
TL;DR: This chapter describes the assay method and purification procedures for chitinase from Verticillium albo-atrum and discusses the properties of the enzyme.
Abstract: Publisher Summary This chapter describes the assay method and purification procedures for chitinase from Verticillium albo-atrum. The fungal enzyme poly-l,4-(2-acetamido-2-deoxy)-β-D-glucoside glycanohydrolase is effective in hydrolyzing chitin and its oligomers chitotetraose, chitotriose, and chitobiose to N-acetylglucosamine (NAG) in the absence of N-acetylglucosaminidase (NAGase). Similarly chitosan is hydrolyzed to glucosamine. The reaction product NAG is determined colorimetrically by a modification of Morgan and Elson's method. The chapter discusses the properties of the enzyme. The enzyme appears stable for several months at 2°. Bovine serum albumin greatly enhances enzyme activity and is essential in the reaction mixture.
TL;DR: The hydrolysis of 3,4-dinitrophenyl tetra N -acetyl β- d -chitotetraoside by intestinal extracts showed a similar profile to that obtained when a cell suspension of Micrococcus lysodeikticus was used as substrate, with an optimum value at pH 5.0–5.5.
Abstract: 1. 1. Chitinolytic activity was studied in the digestive tract of Dover sole Solea solea (L.) using chitin based substrates and synthetic substrates. 2. 2. The initial hydrolysis of chitin appeared to be in the stomach at a pH of 2–3. This activity was detected using glycol chitosan, chitin azure and colloidal chitin as substrates, with minor activity towards 3,4-dinitrophenyl tetra N -acetyl β- d -chitotetraoside in this pH region. 3. 3. This latter substrate was, however, readily hydrolysed at pH 5. At this pH homogenates of the digestive tract of Dover sole also showed activity towards the other substrates tested, with the exception of chitin azure and colloidal chitin. 4. 4. Chitin azure, colloidal chitin and glycol chitosan were hydrolysed at pH values of 8–9 (in addition to their hydrolysis at acid pH values) and it is likely that this alkaline chitinase was originating from intestinal bacteria. 5. 5. The hydrolysis of 3,4-dinitrophenyl tetra N -acetyl β- d -chitotetraoside by intestinal extracts showed a similar profile to that obtained when a cell suspension of Micrococcus lysodeikticus was used as substrate, with an optimum value at pH 5.0–5.5. It is therefore probable that the hydrolysis of substrates at this pH was due to lysozyme.