Showing papers on "Chitin published in 1989"

The degrees of polymerization and N-acetylation of chitosan determine its ability to elicit callose formation in suspension cells and protoplasts of Catharanthus roseus.

Abstract: Partially and fully deacetylated chitosan fragments and oligomers were compared for their potency to elicit formation of the 1.3-β-glucan callose in suspension-cultured cells and protoplasts of Catharanthus roseus (line 385). Chitosan oligomers induced little callose formation, while callose synthesis increased with the degree of polymerization of chitosan up to several thousand corresponding to a molecular mass near 106 Da. At a comparable degree of polymerization, partially N-acetylated chitosan fragments were less effective. Colloidal chitin and chitin oligomers induced only trace callose synthesis in protoplasts. These results indicate that the primary interaction involved the amino groups of chitosan and numerous negative charges at the surface of the plasma membrane with spacing in the nanometer range and occurring regularly over micrometer stretches. Charged phospholipid head-groups may fulfill these requirements. The resulting alteration of membrane fluidity may lead to the changes in ion transport known to be associated with the induction of callose formation.

Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae.

Abstract: Previously, we showed that chitin synthase 2 (Chs2) is required for septum formation in Saccharomyces cerevisiae, whereas chitin synthase 1 (Chs1) does not appear to be an essential enzyme. However, in strains carrying a disrupted CHS1 gene, frequent lysis of buds is observed. Lysis occurs after nuclear separation and appears to result from damage to the cell wall, as indicated by osmotic stabilization and by a approximately 50-nm orifice at the center of the birth scar. Lysis occurs at a low pH and is prevented by buffering the medium above pH 5. A likely candidate for the lytic system is a previously described chitinase that is probably involved in cell separation. The chitinase has a very acidic pH optimum and a location in the periplasmic space that exposes it to external pH. Accordingly, allosamidin, a specific chitinase inhibitor, substantially reduced the number of lysed cells. Because the presence of Chs1 in the cell abolishes lysis, it is concluded that damage to the cell wall is caused by excessive chitinase activity at acidic pH, which can normally be repaired through chitin synthesis by Chs1. The latter emerges as an auxiliary or emergency enzyme. Other experiments suggest that both Chs1 and Chs2 collaborate in the repair synthesis of chitin, whereas Chs1 cannot substitute for Chs2 in septum formation.

Molecular cloning of a cell wall exo-beta-1,3-glucanase from Saccharomyces cerevisiae.

Abstract: A major protein of Saccharomyces cerevisiae cell walls is a 29-kilodalton glycoprotein which shows lectinlike binding to beta-1,3-glucan and chitin. It was solubilized by heating isolated cell walls at 90 degrees C and purified to homogeneity by running two high-pressure liquid chromatography columns. With the sequence information of the N terminus and seven peptides, two oligonucleotides were synthesized and the gene was cloned. Its sequence is similar to those of two plant beta-glucanases, and the protein was shown to possess beta-1,3-exoglucanase activity with laminarin as substrate. Haploid yeast cells contained one copy of the gene (BGL2). Gene disruption did not result in a phenotype.

Degradation and utilization of cellulose and straw by three different anaerobic fungi from the ovine rumen.

Abstract: Three different ruminal fungi, a Neocallimastix sp. (strain LM-1), a Piromonas sp. (strain SM-1), and a Sphaeromonas sp. (strain NM-1), were grown anaerobically in liquid media which contained a suspension of either 1% (wt/vol) purified cellulose or finely milled wheat straw as the source of fermentable carbon. Fungal biomass was estimated by using cell wall chitin or cellular protein in cellulose cultures and chitin in straw cultures. Both strains LM-1 and SM-1 degraded cellulose with a concomitant increase in fungal biomass. Maximum growth of both fungi occurred after incubation for 4 days, and the final yield of protein was the same for both fungi. Cellulose degradation continued after growth ceased. Strain NM-1 failed to grow in the cellulose medium. All three anaerobic fungi grew in the straw-containing medium, and loss of dry weight from the cultures indicated degradation of straw to various degrees (LM-1 greater than SM-1 greater than NM-1). The total fiber component and the cellulose component of the straw were degraded in similar proportions, but the lignin component remained undegraded by any of the fungi. Maximum growth yield on straw occurred after 4 days for strain LM-1 and after 5 days for strains SM-1 and NM-1. The calculated yield of cellular protein for strain LM-1 was twice that of both strains SM-1 and NM-1. The cellular protein yield of strain SM-1 was the same in both cellulose and straw cultures. In contrast to cellulose, straw degradation ceased after the end of the growth phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effect of chitin heparinoids on the lung metastasis of B16-BL6 melanoma.

Abstract: Structure-function studies for the antimetastatic activity of chemically modified chitin heparinoids composed of N-acetyl glucosamine units were performed in an experimental lung metastasis model. 6-O-Sulfated chitin (S-chitin) significantly inhibited the lung tumor colonization in proportion to the degree of sulfation. However, 6-O- and N-sulfated but partially N-deacetylated chitin (S-chitosan), and 6-O-carboxymethylated chitin (CM-chitin) had no effect. 6-O-Sulfated CM-chitin (SCM-chitin), which exhibited fairly low levels of anticoagulant activity, was also more effective than intact heparin. Furthermore, SCM-chitin with a high degree of sulfation (SCM-chitin III) caused a marked decrease of the number of lung tumor colonies in the spontaneous lung metastasis model. These results strongly suggest that 6-O-sulfate and N-acetyl groups in the glucosamine unit were required for the anti-metastatic effect of chitin heparinoids as well as heparin, and SCM-chitin III may be of therapeutic benefit for the prevention of tumor metastasis.

Purification and characterization of chitinase produced by Streptomyces erythraeus.

Abstract: A chitinase was purified from the culture filtrate of Streptomyces erythraeus (SE). The enzyme (SE chitinase) has a molecular weight of 30,000 and pI 3.7, and shows optimal activity at pH 5.0 with an optimal ionic strength of less than 0.2 M NaCl. SE chitinase could hydrolyze chitin and its derivatives, but could not hydrolyze cell walls of Micrococcus lysodeikticus. The substrate specificity of SE chitinase was compared with those of hen egg white (HEW) and SE lysozymes. The binding mode of the chitinase to substrates was investigated using chitooligosaccharides and their derivatives. The results showed that the binding mode of SE chitinase to the substrate is similar to that of HEW and SE lysozymes.

Facile preparation of water-soluble chitin from chitosan.

Abstract: A simple and efficient procedure for the preparation of water-soluble chitin was developed by controlled partial N-acetylation of chitosan. Acetylated chitosans with about 50% substitution prepared in a highly swelled state exhibited high water-solubility, and the procedure proved much superior to the conventional one.

Growth characteristics on cellobiose of three different anaerobic fungi isolated from the ovine rumen.

Abstract: Three morphologically different anaerobic fungi, a Neocallimastix sp. strain (LM-1), a Piromonas sp. strain (SM-1), and a Sphaeromonas sp. strain (NM-1), were isolated from the rumens of sheep. Growth studies were conducted with each isolate in batch cultures by using an anaerobic semidefined medium that lacked ruminal fluid and contained 0.5% cellobiose. Cultures were incubated for periods of up to 10 days, and fungal growth was assessed at regular intervals by dry weight measurements. Samples of fungal biomass were also analyzed for cell-associated protein and, after acid hydrolysis, for chitin as hexosamine. The isolates produced similar yields of dry weight and contained similar amounts of protein. However, strain LM-1 grew at a higher rate and contained less than half the level of chitin compared with the other two isolates. There were high positive correlations between chitin and protein for all three fungi, but comparisons of these parameters with dry weights were affected by the presence of variable amounts of storage carbohydrate. The amount of storage carbohydrate reached maximum levels in strain LM-1 during mid-growth phase and then quickly declined thereafter. When dry weight yields for strain LM-1 were adjusted for changes in storage carbohydrate, high positive correlations were obtained between dry weight and protein or chitin. The storage carbohydrate was probably an alpha-1,4-glucan with alpha-1,6 branches.

Growth responses and lignin production in cell suspensions of Pinus elliottii ‘elicited’ by chitin, chitosan or mycelium of Cronartium quercum f.sp. fusiforme

Abstract: Incubation of suspension-cultured slash pine (Pinus elliotti Engelm.) cells with living mycelial plugs or homogenized, washed and autoclaved mycelium of Cronartium quercum f.sp. fusiforme led to rapid clouding of the medium and visible inhibition of cell growth with only a slight increase in cell mortality. Purified chitin elicited similar changes in cloudiness and growth. Chitin treatment concentrations of up to 1 mg ml-1 showed only slight changes in viability over several days (as with the mycelium and mycelial extract). In contrast, chitosan concentrations of as low as 60 μgml-1 showed cell mortality and browning, with a complete ‘hypersensitive’ response and over 90% cell death occurring within 48 h at between 180 and 270 μ ml-1 chitosan amendment. Phloroglucinol staining showed the presence of lignin-like compounds in the medium and lignin-like compounds were elevated in the cell walls of the chitin, chitosan and mycelium elicited suspension cultures as compared to controls.

β-N-Acetylglucosaminidase from Aspergillus nidulans which degrades chitin oligomers during autolysis

Abstract: A hexosaminidase from autolyzed cultures of Aspergillus nidulans was purified 196 fold and characterized as a beta-N-acetylglucosaminidase (EC 3.2.1.30). The enzyme has a MW of 190000, a pI of 4.3, and optimum pH of 5.0 and is unstable at temperatures above 50 degrees C. The enzyme is a glycoprotein with 19.5% sugars, mannose being the principal component. It binds strongly to chitin. The enzyme hydrolyzes different substrates. The Ki with the competitive inhibitor 2-acetamido-2-deoxy-D-gluconolactone was independent of the substrate used. The enzyme was inhibited by Hg2+, Ag+, acetate and other organic anions. The kinetics of hydrolysis of chitin oligosaccharides from 2 to 6 units was studied by HPLC. This enzyme is an exoenzyme which degraded chitin oligomers gradually with the production of N-acetylglucosamine. The hydrolysis of N-N'-diacetylchitobiose was inhibited non-competitively by glucosamine and N-acetylglucosamine. In mixtures of chitin oligosaccharides, the hydrolysis of chitobiose was competitively inhibited by each of the other oligomers.

Preparation and Release Characteristics of Cisplatin Albumin Microspheres Containing Chitin and Treated with Chitosan

Abstract: Cisplatin (CDDP) containing albumin microspheres and microcapsules incorporating biodegradable macromolecules, chitin and chitosan, were prepared, and their CDDP content and releasing ability and susceptibility to various enzymes were examined. Chitin was incorporated during preparation of the microspheres, while chitosan was used to treat preformed microspheres. CDDP content was remarkably increased by chitin; when chitin was incorporated at a concentration of 1.5%, the CDDP content of the microspheres was found to be 16.2% (1.8 times that with no addition of chitin). CDDP release was suppressed by chitin and chitosan. The 50% CDDP release time was about 1.5 h when no chitin was added, but about 16 h was required when chitin was incorporated into the microspheres at a concentration of 1.5%. Chitin and chitosan suppressed the decomposition by protease. The microspheres treated with 70% deacetylated chitosan showed the greatest susceptibility to lysozyme. In conclusion, CDDP release can be controlled by the use of chitin or chitosan, and the microspheres should show no immunogenicity in vivo because of their susceptibility to lysozyme.

Endochitinase from Aspergillus nidulans implicated in the autolysis of its cell wall

Abstract: An endochitinase from centrifuged autolyzed cultures of Aspergillus nidulans has been purified 100 times. The enzyme has Mw 27,000, pI of 4.8 units, pH optimum around 5 pH units. It is unstable at temperature greater than 70 degrees C and does not have a cation requirement. It is inhibited by Hg2+, Cu2+, Ca2+ and Ag+ and it does not have muramidase activity. The enzyme depolymerizes chitin rapidly with production of high molecular weight polysaccharides, and then slowly degrades these with production of N,N'-diacetylchitobiose. The enzyme hydrolyzes N,N',N''-triacetylchitotriose with production of N,N'-diacetylchitobiose and N-acetylglucosamine and this hydrolysis is inhibited by other chitin oligomers and N-acetylglucosamine. This enzyme hydrolyzes in the same way the chitin obtained from the cell wall of Aspergillus nidulans.

Purification and some properties of two forms of chitinase from mycelial cells of Mucor rouxii.

Abstract: Chitinase activity was measured in extracts of mycelial cells of Mucor rouxii as a function of the culture age. There was a peak of specific activity at the mid-exponential phase of growth (10 h), which paralleled chitin synthase activity. An additional peak of chitinase with higher specific activity was detected in 4 h cultures, which coincided with the onset of germination. Purification of chitinase activities from the cytoplasm revealed two enzymes, I and II, with different molecular mass and ionic charge. Antibodies induced with chitinase I did not cross-react with chitnase II. Both enzymes digested nascent chitin preferentially over preformed chitin, yielding diacetylchitobiose as the sole product of hydrolysis.

Modified cellulose for biocompatible dialysis membranes IV and process for preparation thereof

Abstract: A modified cellulose for biocompatible dialysis membranes having a structure represented by the formula ##STR1## wherein cell is unmodified cellulose or chitin, in each case without hydroxyl groups, s=3 in the case of cellulose and s=2 in the case of chitin, R' is CH 3 and/or C 2 H 5 and/or C 3 H 7 , X denotes specified functional groups, m=0.75 to 2.85, and x=0.005 to 2.10. A process for preparation of the cellulose derivatives is also disclosed.

High strength chitosan fibers and fabrics thereof

Abstract: High strength chitosan fibers from chitin and chitosan derivatives and the process for making these fibers are disclosed.

Alterations in the vesicular pattern and wall growth ofPhycomyces induced by the calcium ionophore A 23187

Abstract: Hyphal elongation, chitin synthesis in vivo, and invertase secretion inPhycomyces blakesleeanus were all inhibited almost instantly by the addition of 5–10 μM calcium ionophore A 23187. Protein biosynthesis was inhibited in these conditions by 30–50%. The ionophore did not affect cell respiration for at least 40 min. Effect on chitin biosynthesis was not due to alterations of the chitin synthetase levels or its activity; nor to impairement in GlcNAc metabolism. In drug-treated cells the number of apical vesicles was severely reduced even at very short periods of incubation, and these low numbers remained constant for at least 60 min of incubation with the ionophore. We suggest that the ionophore collapses the cellular calcium gradient and/or interferes with the normal electrical transhyphal current. As a consequence, formation and migration of apical vesicles are inhibited. These results are further evidence of the role of vesicles in fungal tip growth and exhibit the fact that active chitin synthetase is short-lived in vivo demanding its continuous supply by chitosomes to the cell surface.

Method to detect fungi and yeasts

Abstract: A method for detecting organisms such as fungi and yeast in a sample attached to a solid phase, by detecting the presence of chitin in using lectin or anti-chitin antibodies.

Chitin degradation by clostridium sp strain-9.1 in mixed cultures with saccharolytic and sulfate-reducing bacteria

Abstract: The fermentation of chitin was studied in pure and cocultures of the chitinolytic Clostridium strain 9.1 and various non-hydrolytic sugar-fermenting and sulfate-reducing bacteria. A 5- to 8-fold enhancement of the rate of chitin degradation was observed, which was not due to the alleviation of inhibition of the chitinolytic enzyme system by polymer hydrolysis products. This was concluded from the observation that rates of chitinolysis and fermentation were unaffected by the addition of N -acetylglucosamine (NAG) or NAG-oligomers to pure cultures of strain 9.1, and from the absence of an unequivocal relation between the ability of a secondary bacterium to consume potentially inhibitory hydrolysis products and its capacity to stimulate chitin degradation. The acceleration of chitin fermentation in the presence of sugar-fermenting bacteria was the result of a release by these secondary populations of growth factors essential to strain 9.1. These factors comprised a high molecular, thioredoxin-like compound responsible for enhanced chitinolytic activity [10], and various low molecular compounds necessary for optimal growth. The sulfate reducers (except Desulfovibrio sp. strain 20028) released primarily the high molecular growth factor in coculture with strain 9.1. NAG-fermenting bacteria consumed approximately 10% of the hydrolysis products, whereas species capable of utilizing both mono- and oligomeric sugars fermented at least 50% of the sugars produced by strain 9.1. Nevertheless, the rate of chitinolysis in these cocultures proceeded at very similar rates. The observed interactions between Clostridium sp. strain 9.1 and the secondary populations are discussed in relation to the results from studies on mixed culture fermentations of cellulosic substrates reported in the literature.

Immobilization and purification of fusion proteins using chitin-binding ability

Abstract: A gene fusion construction encoding a protein of interest and the chitin binding domain of a chitinase enzyme and the encoded fusion protein, which is the protein of interest and a protein capable of binding chitin. A plasmid vector containing the gene fusion construction is also described. The vector is used to transform host cells, which produce and secrete the recombinant fusion protein. The fusion protein is isolated by binding to chitin through its chitin-binding domain. The present method uses chitin to bind the fusion protein through its chitin-binding domain, and thereby allows immobilization and/or purification of the protein of interest, using known techniques.

Chitin and β(1–3) glucan synthases in the protoplastic entomophthorales

Abstract: β(1–3) glucan and chitin synthases were studied in spontaneously produced protoplasts and in the mycelium (hyphal body) of the entomopathogenic Entomophthorale species Entomophaga aulicae, Conidiobolus obscurus and Entomophthora muscae. The absence of wall in protoplasts was correlated to an absence of chitin synthase and to a very low β(1–3) glucan synthase activity, whereas these two polysaccharide synthases were present and active in the walled hyphal bodies. Physicochemical properties of chitin and β(1–3) glucan synthases such as localization, optimum pH and temperature, activation by disaccharides and proteases were similar to those found in other fungi unable to spontaneously produce protoplasts and could not be related to the ability for protoplastic Entomophthorale species to produce and proliferate under a protoplast form. The absence or the low chitin and glucan synthase activites in Entomophthorale protoplasts was not due to an absence of proteolytic activation of the enzyme. However, all protoplast fractions contained inhibitory substances of glucan and chitin synthase activities. These inhibitors were stable and specific of the protoplast stage. They were not glucanase nor chitinase. These results suggest that the absence of wall synthesis in Entomophthorale protoplasts is due to a continuous inhibition of β(1–3) glucan and chitin synthase activities by intracellular compounds and also for glucan synthase by protoplast medium constituents such as NaCl and fetal calf serum.

A Development of Food Preservative with the Waste of Crab Processing

Abstract: This experiment was carried out to develop the preparation method of chitosan which has strong antimicrobial activity, and also tried to investigate as a natural food preservative with this chitosan. The antimicrobial activity of chitosan was the strongest when deacetylation of chitin was conducted at for 8 hours with sodium hydroxide. The growth of Escherichia coil was completely inhibited by adding this low molecular weight chitosan (M. W, 35,000) at the level of concentration of 75ppm to the medium. The antimicrobial activity was strong enough against such Gram positive bacteria as Staphylococcus sp. and Bacillus sp.. The growth of these strains was inhibited by the concentration of 50ppm but it was varied in its kinds against Gram negative bacteria. The concentration of chitosan re-quired for growth inhibition of microorganisms was 100ppm against Pseudomonas sp. and Vibrio sp., 2,000ppm against Salmonella sp.. The growth of Saccharomyces sp. was inhibited by the concentration of 100ppm, but Hansenula sp., Aspergillus sp., Penicillium sp. and Mu-cor sp. did not inhibited by even more than the concentration of 5,000ppm. The shelf life of Mulkimchi (pickle type Kimchi), containing chitosan was 10 days longer than control stored at .

Studies on graft copolymerization onto cellulose derivatives. XXIX. Photo‐induced graft copolymerization of methyl methacrylate onto chitin and oxychitin

Abstract: Graft copolymerization of methyl methacrylate (MMA) onto chitin and oxidized chitin was carried out by a noncatalytic photo-induced and a photo-sensitized method. The isolation method of grafted chains without a decrease in their degree of polymerization (DP) by sulfuric acid hydrolysis and the effects of some factors on the conversion and the grafted chain length have been studied. In case of the photo-induced graft copolymerization, if a small amount of dimethylformaldehyde (DMF) is added in the polymerization system, the induction period is shortened and the degree of grafting and the apparent number of grafted chains increase. The degree of grafting and the apparent number of grafted chains of the oxidized chitin containing a small amount of CO groups are greater than those of the untreated chitin, but a further increase in CO groups decreases these values. In the presence of this two species of the chitin samples, the polymerization reaction was carried out as functions of monomer and chitin concentrations. From these results, the mechanisms of the photo-induced graft copolymerization were discussed. It is also clear that the conversion and the apparent number of grafted chains by the noncatalytic photo-induced method are generally larger than those by the photo-sensitized method, and the grafting activity with hydrogen peroxide (HPO) is higher than that with azobisisobutyronitrile (AIBN).

Role of luminous bacteria in chitin degradation in the intestine of fish

Abstract: Chitinolytic activity of three luminous bacterial species viz.,Vibrio harveyi, V. fischeri andPhotobacterium leiognathi, was estimated. The stomach of the fishTachysurus arius which harbours luminous bacteria also possesses chitinase activity. The magnitude of non-bacterial chitinase activity appeared to exceed that of the bacterial origin as evidenced by the enzyme activity detected in fish tested with antibiotics. In fishes which lack an indigenous chitinase, the microbial chitinase is likely to play an important role in chitin digestion.