The control of chitin deposition by ecdysterone in larvae of Bombyx mori

The effect of ecdysterone on the deposition of chitin in Bombyx larvae was examined. The chitin content in the abdomen decreased following hormone treatment to a value half that of the controls. Studies with ¹⁴C-glucose revealed that whereas controls exhibited a gradual decrease in the rate of ¹⁴C-glucose incorporation into chitin, the administration of ecdysterone resulted in a rapid fall in the rate of incorporation followed by a rise until ecdysis occurred. Chitin catabolism was estimated at 0·11 mg chitin/hr, based on the chitin content and incorporation rates. A dual rôle is indicated for ecdysterone in chitin metabolism, namely the activation of both synthetic and lytic systems.     

Defects in Assembly of the Extracellular Matrix Are Responsible for Altered Morphogenesis of a Candida albicans phr1 Mutant

Analysis of Candida albicans cells using antibodies directed against Gas1p/Ggp1p, Saccharomyces cerevisiae homolog of Phr1p, revealed that Phr1p is a glycoprotein of about 88 kDa whose accumulation increases with the rise of external pH. This polypeptide is present both in the yeast form and during germ tube induction. In the Phr1⁻ cells at pH 8 the solubility of glucans in alkali is greatly affected. In the parental strain the alkali-soluble/-insoluble glucan ratio shows a 50% decrease at pH 8 with respect to pH 4.5, whereas in the null mutant it is unchanged, indicating the lack of a polymer cross-linker activity induced by the rise of pH. The mutant has a sixfold increase in chitin level and is hypersensitive to calcofluor. Consistently with a role of chitin in strengthening the cell wall, Phr1⁻ cells are more sensitive to nikkomycin Z than the parental strain.     

Quantitative isolation of native chitin from resistant structures of Sordaria and Ascaris species

A procedure for the quantitative isolation of native chitin from resistant biological structures is developed utilizing ozone and neutralized hydrogen peroxide ethylenediaminetetraacetic acid. These reagents permit extraction of the chitinous component at a neutral pH without any preliminary cell disruption or extensive fractionation, thereby conserving the cell wall integrity and structure. The resulting product is essentially free of protein and is greatly enriched in chitin content, the exact purity of which depends upon the nature of the particular chitin source. The chitin polymer is recovered in a highly N-acetylated form and is readily identified with a standard chitin reference by physical characterization, infrared spectroscopy, enzymatic hydrolysis, and glucosamine assay.     

Inhibitory effect of chitinases isolated from Semillon grapes (Vitis vinifera) on growth of grapevine pathogens

Characteristics and antifungal activity of chitinases in Semillon grapes were investigated. Chitinases were isolated from the juice of Semillon grapes by chitin affinity chromatography. Native and SDS-PAGE analyses of the fraction showing chitin affinity (active fraction) demonstrated only the presence of protein bands of chitinases. Three types of class IV chitinases (chi-1a, chi-1b and chi-2) were purified from the active fraction. These chitinases actively hydrolyzed chitin under acidic conditions (pH 4.0–4.5). The isoelectric points and the molecular weights of chi-1a, chi-1b and chi-2 were 4.73, 4.60, and 7.87, and 32.1 kDa, 31.6 kDa, and 29.0 kDa, respectively. The active fraction was found to inhibit Botrytis cinerea mycelial growth and the inhibitory effect was due to the activity of chitinases. The active fraction inhibited twenty strains of B. cinerea collected from the experimental vineyard. The effect of chitinases was enhanced in media containing more than 20% sugar. When the active fraction was tested on Glomerella cingulata, the growth inhibitory effect observed was markedly less than that seen on B. cinerea.     

Some properties of chitinase produced by a potent Aspergillus carneus strain

Summary Seven fungal isolates characterized by high chitinolytic activity were isolated from soil and identified. Aspergillus carneus in a 7-day-old shaken culture was the most promising chitinase producer. The use of chitin as a carbon source favoured production of extracellular chitinase enzymes. Maximum chitinase activity was reached at 10 g chitin/1. An initial pH value of the culture medium of 5.0 gave the highest chitinolytic activity. Some properties of the crude enzyme produced by A. carneus were studied. Maximal enzyme activity was reached at pH 4.5 and 40° C after 30 min. Thermal treatments at 70° C and pH 4.5 had the most adverse effect on enzyme activity.Offprint requests to: M. A. Abd El-Naby     

Chitinolytic enzymes from<Emphasis Type="Italic">Clostridium aminovalericum</Emphasis>: Activity screening and purification

A strain isolated from the feces of takin was identified as Clostridium aminovalericum. In response to various types of chitin used as growth substrates, the bacterium produced a complete array of chitinolytic enzymes: chitinase ('endochitinase'), exochitinase, beta-N-acetylglucosaminidase, chitosanase and chitin deacetylase. The highest activities of chitinase (536 pkat/mL) and exochitinase (747 pkat/mL) were induced by colloidal chitin. Fungal chitin also induced high levels of these enzymes (463 pkat/mL and 502 pkat/mL, respectively). Crab shell chitin was the best inducer of chitosanase activity (232 pkat/mL). The chitinolytic enzymes of this strain were separated from culture filtrate by ion-exchange chromatography on the carboxylic sorbent Polygran 27. At pH 4.5, some isoforms of the chitinolytic enzymes (30% of total enzyme activity) did not bind to Polygran 27. The enzymes were eluted under a stepwise pH gradient (pH 5-8) in 0.1 mol/L phosphate buffer. At merely acidic pH (4.5-5.5), the adsorbed enzymes were co-eluted. However, at pH close to neutral values, the peaks of highly purified isoforms of exochitinases and chitinases were isolated. The protein and enzyme recovery reached 90%.     

Bacterial Chitinolytic Communities Respond to Chitin and pH Alteration in Soil

Chitin amendment is a promising soil management strategy that may enhance the suppressiveness of soil toward plant pathogens. However, we understand very little of the effects of added chitin, including the putative successions that take place in the degradative process. We performed an experiment in moderately acid soil in which the level of chitin, next to the pH, was altered. Examination of chitinase activities revealed fast responses to the added crude chitin, with peaks of enzymatic activity occurring on day 7. PCR-denaturing gradient gel electrophoresis (DGGE)-based analyses of 16S rRNA and chiA genes showed structural changes of the phylogenetically and functionally based bacterial communities following chitin addition and pH alteration. Pyrosequencing analysis indicated (i) that the diversity of chiA gene types in soil is enormous and (i) that different chiA gene types are selected by the addition of chitin at different prevailing soil pH values. Interestingly, a major role of Gram-negative bacteria versus a minor one of Actinobacteria in the immediate response to the added chitin (based on 16S rRNA gene abundance and chiA gene types) was indicated. The results of this study enhance our understanding of the response of the soil bacterial communities to chitin and are of use for both the understanding of soil suppressiveness and the possible mining of soil for novel enzymes.     

Purification and properties of chitinase from Streptomyces cinereoruber

A chitinase (EC 3.2.1.14) was purified from the culture filtrate of Streptomyces cinereoruber, selected as a microorganism which produces enzymes lysing Aspergillus niger cell wall, by fractional precipitation with ammonium sulfate and column chromatographies on DEAE-cellulose, Sephadex G-100 and CM-Sephadex C-50. The final preparation was homogenous in polyacrylamide gel disc electrophoresis. The molecular weight of the enzyme was about 19,000 daltons and its isoelectric point was pH 8.6. The optimum pH and temperature for chitinase activity were 4.5 and at 50°C, respectively. The enzyme was stable in the pH range from 4.0 to 10.0. The activity was inhibited by Ag⁺, Hg⁺, Hg²⁺ and p-chloromercuribenzoate. Paper chromatographic analysis demonstrated that the hydrolytic products of colloidal chitin and chitotriose with the enzyme were N-acetylglucosamine and chitobiose. The lysis of A. niger cell wall with the enzyme is discussed.     

Possible physiological mechanisms for the differential susceptibility of two forest Lepidoptera to diflubenzuron

In an attempt to explain the physiological mechanisms for the differential susceptibility of insects to the chitin synthesis inhibitor, diflubenzuron, chitin content, chitin synthesis, and retention of ingested ¹⁴C-diflubenzuron in two forest Lepidoptera were investigated. The spruce budworm, Choristoneura fumiferana, a refractory species, had less chitin and retained less of the ingested material than the forest tent caterpillar, Malacosoma disstria, a species highly sensitive to diflubenzuron. No difference in the chitin synthesis pattern during the 6th stadium was observed in the two species. It is concluded that the primary reasons for the increased susceptibility of the forest tent caterpillar to this compound was the increased retention of ingested diflubenzuron and, to a lesser extent, the increased chitin content.     

Culture of Vibrio cholerae in presence of shrimp and crab chitin

In the culture of Vibrio cholerae in saline water containing purified chitin prepared from fresh shrimp and crab shells, it was determined that the solubility of chitin in salin water is influenced more by the salinity than by the pH, with the optimum being pH 8·0 and salinity 15 o/oo. The stimulation of growth by freshly extracted chitins and commercially available preparations was similar. All chitin preparations stimulated growth somewhat less than did alkaline peptone broth (a commonly used culture medium). All chitin sources also stimulated the production of cholera toxin by toxigenic strains to about the same extent as did alkaline peptone broth. The strains were found to be very low toxin producers in filtered bay water alone, thus indicating the need for some nutrient source for this activity.     

Secondary cell wall formation in Cryptococcus neoformans as a rescue mechanism against acid-induced autolysis

Growth of the opportunistic yeast pathogen Cryptococcus neoformans in a synthetic medium containing yeast nitrogen base and 1.0–3.0% glucose is accompanied by spontaneous acidification of the medium, with its pH decreasing from the initial 5.5 to around 2.5 in the stationary phase. During the transition from the late exponential to the stationary phase of growth, many cells died as a consequence of autolytic erosion of their cell walls. Simultaneously, there was an increase in an ecto-glucanase active towards β-1,3-glucan and having a pH optimum between pH 3.0 and 3.5. As a response to cell wall degradation, some cells developed an unusual survival strategy by forming 'secondary' cell walls underneath the original ones. Electron microscopy revealed that the secondary cell walls were thicker than the primary ones, exposing bundles of polysaccharide microfibrils only partially masked by an amorphous cell wall matrix on their surfaces. The cells bearing secondary cell walls had a three to five times higher content of the alkali-insoluble cell wall polysaccharides glucan and chitin, and their chitin/glucan ratio was about twofold higher than in cells from the logarithmic phase of growth. The cell lysis and the formation of the secondary cell walls could be suppressed by buffering the growth medium between pH 4.5 and 6.5.     

Serratia marcescens chitinase: One-step purification and use for the determination of chitin

The extracellular chitinase produced by Serratia marcescens was obtained in highly purified form by adsorption-digestion on chitin. After gel electrophoresis in a nondenaturing system, the purified preparation exhibited two major protein bands that coincided with enzymatic activity. A study of the enzyme properties showed its suitability for the analysis of chitin. Thus, the chitinase exhibited excellent stability, a wide pH optimum, and linear kinetics over a much greater range than similar enzymes from other sources. The major product of chitin hydrolysis was chitobiose, which was slowly converted into free N-acetylglucosamine by traces of β-N-acetylglucosaminidase present in the purified preparation. The preparation was free from other polysaccharide hydrolases. Experiments with radiolabeled yeast cell walls showed that the chitinase was able to degrade wall chitin completely and specifically.     

Preparation of fermentation-processed chitin and its application in chitinase affinity adsorption

A fermentation approach utilizing Paenibacillus sp. to process chitin was developed. The chitin obtained from this process is called fermentation-processed chitin (FPC), and it was further investigated with chitinase affinity adsorption studies together with three other adsorbents, i.e. crab shell chitin, colloid chitin, and enzyme-processed chitin. The results showed that FPC had the highest chitinase adsorption capacity. Under 15 °C and pH 5.0, FPC exhibited an optimal chitinase adsorption capacity of 85.9 U/g, which was 61.9% higher than that of the colloidal chitin. With 0.02 M acetic acid as the eluent, a purification-fold of 10.3 with 97% chitinase recovery was obtained. The results of surface morphology studies indicated that the FPC surface was modified to a fiber-like structure with deep pores. In comparison with the surface morphology of enzyme-processed chitin and colloidal chitin, it is inferred that the enhanced adsorption capacity of FPC for chitinase is attributed to both the effects of chitinase hydrolysis and the bacterial modification.     

Bacterial chitin utilisation at extremely haloalkaline conditions

Chitin is produced in large amounts in hypersaline habitats with neutral pH due to the high biomass production of brine shrimp Artemia. Recently, a high abundance of Artemia was also noticed in hypersaline soda lakes in the Kulunda Steppe (Altai, Russia), which prompted us to survey the possibility of microbial chitin utilization at extremely haloalkaline conditions in soda brines. Most active chitin utilisation-supporting microbial growth was found at anaerobic conditions at pH 10 and up to 3.5?M total Na⁺. At aerobic conditions, the degradation of chitin was slower, mostly incomplete and active at <2?M total Na⁺, although very slow partial degradation was possible up to 4?M Na⁺. Anaerobic enrichments at pH 10 yielded two different groups of obligately haloalkaliphilic fermentative anaerobes, exclusively specialized to utilise insoluble chitin as the only growth substrate. One group was represented by a single strain growing at moderate salinity, and another comprised multiple isolates growing up to 3.5?M Na⁺. These groups represent two novel bacterial phyla not closely related to any other cultured bacteria. Aerobic enrichments from the lake sediments were dominated by several obligately haloalkaliphilic members of the genus Marinimicrobium in the Gammaproteobacteria. They were less specialised than the anaerobes and grew with chitin and its monomer and oligomers at a pH of 10 up to 2.5?M Na⁺. Furthermore, several strains of haloalkaliphilic Gram-positive chitinolytics belonging to bacilli and actinobacteria were isolated from soda lake sediments and surrounding soda soils. In general, the results indicate the presence of an active and diverse haloalkaliphilic chitinolytic microbial community in hypersaline soda habitats.     

Purification and characterization of an extracellular chitosanase produced by Amycolatopsis sp. CsO-2

Extracellular chitosanase produced by Amycolatopsis sp. CsO-2 was purified to homogeneity by precipitation with ammonium sulfate followed by cation exchange chromatography. The molecular weight of the chitosanase was estimated to be about 27,000 using SDS-polyacrylamide gel electrophoresis and gel filtration. The maximum velocity of chitosan degradation by the enzyme was attained at 55°C when the pH was maintained at 5.3. The enzyme was stable over a temperature range of 0–50°C and a pH range of 4.5–6.0. About 50% of the initial activity remained after heating at 100°C for 10 min, indicating a thermostable nature of the enzyme. The isoelectric point of the enzyme was about 8.8. The enzyme degraded chitosan with a range of deacetylation degree from 70% to 100%, but not chitin or CM-cellulose. The most susceptible substrate was 100% deacetylated chitosan. The enzyme degraded glucosamine tetramer to dimer, and pentamer to dimer and trimer, but did not hydrolyze glucosamine dimer and trimer.     

Characterization of Chitinase Produced by the Alkaliphilic <Emphasis Type="Italic">Bacillus mannanilyticus</Emphasis> IB-OR17 B1 Strain

The paper reports on the isolation of an extracellular chitinase produced by the alkaliphilic Bacillus mannanilyticus IB-OR17 B1 strain grown in media containing crab shell and bee chitin at a pH of 8–11. The enzyme was 860-fold purified by ultrafiltration and chitin sorption. The molecular weight of the purified chitinase was shown by denaturing electrophoresis to be 56 kDa. The enzyme showed maximum activity at a pH of 7.5–8.0 and 65°C and was stable within a pH range of 3.5–10.5 and temperature range of 75–85°C. With colloidal chitin as substrate, the kinetic characteristics of the chitinase were determined as follows: K_M ~ 1.32 mg/mL and V_max ~ 5.05 μM min^–1. N-acetyl-D-glucosamine and its dimer were the main products of enzymatic chitin cleavage, while the trisaccharide was detected just in minor quantities. The chitinase actively hydrolyzed p-nitrophenyl-GlcNAc₂ according to the exo-mechanism of substrate hydrolysis characteristic of chitobiosidases.     

Conversion of the enzymatic hydrolysate of shellfish waste chitin to single-cell protein

The conversion of the enzymatic hydrolysate of shellfish chitin waste to single-cell protein was investigated as part of a comprehensive waste treatment program. Forty-two yeasts were screened for ability to assimilate the monomer of chitin, N-acetylglucosamine, which has been shown to be the sole product of enzymatic hydrolysis of chitin. The Yeast Pichia Kudriavzevii was selected for study, based on ability to grow at high temperature (37°C and above), low pH (4.0 ± 0.5), and in a nutritionally simple medium. Growth rates of P. kudriavzevii were similar on N-acetylglucosamine and on the chitin hydrolysate. Dependencies of specific growth rate on temperature, pH, medium composition, and oxygen tension were studied. The variations of yield, protein content, and total nucleic acid content with the specific growth rate were evaluated. The amino acid distribution of the protein of P. kudriavzevii was obtained.     

High-yield production of a chitinase from Aeromonas veronii B565 as a potential feed supplement for warm-water aquaculture

Chitin, present in crustacean shells, insects, and fungi, is the second most plentiful natural organic fiber after wood. To effectively use chitin in a cost-saving and environmentally friendly way in aquaculture, crustacean shells (e.g., shrimp-shell meal) are supplemented into aquafeed after degradation by chemical methods. Herein, we describe a chitinase from Aeromonas veronii B565, designated ChiB565, which potently degrades shrimp-shell chitin and resists proteolysis. We isolated recombinant ChiB565 of the expected molecular mass in large yield from Pichia pastoris. ChiB565 is optimally active at pH 5.0 and 50 °C and stable between pH 4.5 and 9.0 at 50 °C and below. Compared with the commercial chitinase C-6137, which cannot degrade shrimp-shell chitin, ChiB565 hydrolyzes shrimp-shell chitin in addition to colloidal chitin, powdered chitin, and β-1,3-1,4-glucan. The optimal enzyme concentration and reaction time for in vitro degradation of 0.1 g of powdered shrimp shell are 30 U of ChiB565 and 3 h, respectively. A synergistic protein-release effect occurred when ChiB565 and trypsin were incubated in vitro with shrimp shells. Tilapia were fed an experimental diet containing 5 % (w/w) shrimp bran and 16.2 U/kg ChiB565, which significantly improved growth and feed conversion compared with a control diet lacking ChiB565. Dietary ChiB565 enhanced nitrogen digestibility and downregulated intestinal IL-1β expression. The immunologically relevant protective effects of dietary ChiB565 were also observed for 2 to 3 days following exposure to pathogenic Aeromonas hydrophila.     

Purification and characterization of a chitinase from Serratia proteamaculans

A chitinase gene from Serratia proteamaculans 18A1 was cloned, sequenced, and expressed in Escherichia coli M15. Recombinant enzyme (ChiA) was purified by Ni-NTA affinity column chromatography. The ChiA gene contains an open reading frame (ORF), encoding an endochitinase with a deduced molecular weight 60 kDa and predicted isoelectric point of 6.35. Comparison of ChiA with other chitinases revealed a modular structure containing an N-terminal PKD-domain, a family 18 catalytic domain and a C-terminal putative chitin-binding domain. Turn over rate (K _cat) of the enzyme was determined using colloidal chitin (49.71 ± 1.15 S^?1) and crystalline β-chitin (17.20 ± 0.83 S^?1) as substrates. The purified enzyme was active over a broad range of pH (pH 4.5–9.0) and temperature (4–70°C) with a peak activity at pH 5.5 and 55°C. However, enzyme activity was found to be stable up to 45°C for longer incubation periods. Purified enzyme was shown to inhibit fungal spore germination and hyphal growth of pathogenic fungi Fusarium oxysporum and Aspergillus niger.