Remazol Brilliant Violet Research Articles

Cassia angustifolia seed gum as an effective natural coagulant for decolourisation of dye solutions

Owing to economic advantages in addition to the potential for improved process robustness and to reduce the pollution load on the environment, particularly in water treatment applications in developing countries, an investigation with biodegradable and eco-friendly naturally occurring seed gum as coagulant was undertaken. Naturally occurring Cassia angustifolia (CA) seed gum was evaluated against the chemical coagulant polyaluminium chloride (PAC) for its coagulation ability to remove colour from synthetic dye solutions. Three groups of dyes: Acid Sendula Red, Direct Kahi Green and Reactive Remazol Brilliant Violet were chosen for the case study. For optimum results the variables studied were pH and dosage. CA was found to be a good working substitute alone or in conjunction with a very low dose of PAC for decolourisation of acid and direct, but not for reactive dye solutions. The performance of PAC was better with direct dye and that of CA was better with that of acid dye.

Purification and Characterization of Chitinases from the Nematophagous Fungi Verticillium chlamydosporium and V. suchlasporium

Culture filtrates of the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium growing on colloidal chitin showed increasing chitinolytic activity and production of two (32- and 43-kDa) main proteins. Maximum activity was found 18–20 days after inoculation, but V. suchlasporium always displayed higher activity. Zymography of such filtrates on carboxymethyl–chitin–Remazol brilliant violet 5R/acrylamide gels showed five bands of substrate degradation for V. suchlasporium and three for V. chlamydosporium. Filtrates with maximum activity were chromatographed on macroporous cross-linked chitin affinity matrix, showing a peak of main (50–60%) activity, which only contained a 43-kDa protein for both fungi. Zymography and colloidal chitin degradation showed that it was a single endochitinase (CHI43) with optimum pH range of 5.2–5.7. The main isoforms had pIs of 7.6 for V. suchlasporium and 7.9 for V. chlamydosporium. Eggs of the nematode Globodera pallida treated with CHI43 and the serine protease P32 from V. suchlasporium alone or in combination showed surface damage in comparison with controls when examined by scanning electron microscopy.

Enzyme activity assays for nutrient solutions from closed irrigation systems

Exo-enzymes degrading cell wall components of fungal pathogens may play a role in the biological control of plant diseases in hydroponic systems. Therefore, protease, cellulase and chitinase are of special interest. The main objective of this work was the development of enzyme assays suitable for the conditions found in nutrient solutions. Furthermore, inducibility of enzyme activities by amendment of organic substrates to nutrient solutions and the effect of storage on enzyme activities in nutrient solutions were investigated. Enzyme tests based on the rate of release of soluble dye-labeled fragments of chromogenic substrates were adapted for nutrient solutions and were evaluated as suitable for the purpose. Using Hide-Remazol Brilliant Blue R as substrate for protein degrading enzymes, a stable activity of protease for up to 16 h was found. In order to detect the activity of chitinolytic enzymes, Carboxymethyl-Chitin Remazol Brilliant Violet was utilized as substrate. Results indicated good performance of the test, although chitinase activity was not observed in the beginning but later in the crop season. Furthermore, an approach to detect cellulase activity using Carboxymethyl-Cellulose Remazol Brilliant Blue as substrate yielded satisfying results. Amendment of nutrient solutions with chitin and cellulose (1%) incubated for 3 days led to significantly increased activity of chitinase and cellulase, respectively. For all of the studied enzymes, storage at +2 °C did not affect the activity for at least 1 week. In contrast, storage of samples of nutrient solution at room temperature led to a rapid decrease in activity of chitinase and cellulase, whereas protease activity was negatively affected by freezing (−26 °C) the nutrient solution.

Effect of some operational parameters on textile dye biodegradation in a sequential batch reactor

The combination of anaerobic and aerobic periods in the operation cycle of a Sequencing Batch Reactor (SBR) was chosen to study biological color removal from simulated textile effluents containing reactive, sulfonated, monoazo and diazo dyes, respectively, Remazol Brilliant Violet 5R and Remazol Black B. 90% color removal was obtained for the violet dye in a 24-h cycle with a Sludge Retention Time (SRT) of 15 days and an aerated reaction phase of 10 h. For the black dye only 75% color removal was achieved with the same operational conditions and no improvement was observed with the increase of the SRT to 20 days. For the violet dye a reduction of the color removal values from 90 to 75% was observed with the increase of the aerated reaction phase from 10 to 12 h. However, this increase did not promote the aerobic biodegradation of the produced aromatic amines. Abiotic tests were performed with sterilized SBR samples and no color removal was observed in cell-free supernatants. However color removal values of 30 and 12% were observed in the presence of sterilized cells and supernatants with violet and black dye, respectively and could be attributed to the presence of active reducing principles in the sterilized samples.

A Bacillus amyloliquefaciens ChbB protein binds beta- and alpha-chitin and has homologues in related strains.

A small (19.8 kDa) protein was identified in Bacillus amyloliquefaciens ALKO 2718 cultures during growth in the presence of yeast extract and chitin, but not with glucose. The protein targets beta-chitin best, then alpha-chitin, but barely any other polysaccharide. This described chitin-binding protein (ChbB) is the first of its type from a Bacillus strain and cross-reacts with antibodies raised against the Streptomyces alpha-chitin-binding protein CHB1. Using reverse genetics, the chromosomal chbB gene of strain ALKO 2718 was identified, cloned and sequenced. ChbB shares several motifs with the alpha-chitin-binding proteins CHB1 and CHB2 of Streptomyces and CBP21 of Serratia marcescens predominantly targeting beta-chitin. Synthesis was repressed by glucose and the presence of cre boxes suggests catabolite control. Using PCR, Southern hybridization and anti-ChbB antibodies, the presence of a chbB gene, as well as of a ChbB protein homologue, was ascertained in several tested B. amyloliquefaciens strains, but not in Bacillus subtilis 168. Contrary to B. subtilis 168, all B. amyloliquefaciens strains secreted varying amounts of enzymic activity, degrading carboxymethyl chitin coupled with Remazol brilliant violet.

Assessment of chitin decomposer diversity within an upland grassland.

The breakdown of chitin within an acidic upland grassland was studied. The aim was to provide a molecular characterisation of microorganisms involved in chitin degradation in the soil using soil microcosms and buried litter bags containing chitin. The investigation involved an examination of the effects of liming on the microbial communities within the soil and their chitinolytic activity. Microcosm experiments were designed to study the influence of lime and chitin enrichment on the grassland soil bacterial community ex situ under controlled environmental conditions. Bacterial and actinomycete counts were determined and total community DNA was extracted from the microcosms and from chitin bags buried at the experimental site. PCR based on specific 16S rRNA target sequences provided products for DGGE analysis to determine the structure of bacterial and actinomycete communities. Chitinase activity was assessed spectrophotometrically using chitin labelled with remazol brilliant violet. Both liming and chitin amendment increased bacterial and actinomycete viable counts and the chitinase activity. DGGE band patterns confirmed changes in bacterial populations under the influence of both treatments. PCR products amplified from DNA isolated from chitin bags were cloned and sequenced. Only a few matched known species but a prominent coloniser of chitin proved to be Stenotrophomonas maltophilia.

Reactive textile dye colour removal in a sequencing batch reactor

Two sequencing batch reactors (SBRs) with sequenced anaerobic/aerobic phases were used to study biological colour removal from a simulated cotton textile effluent containing an azo reactive dye. One of the reactors was daily fed with Remazol Brilliant Violet 5R dye and the other was used as control. When operating with a sludge retention time (SRT) of 15 days the total COD removal was around 80%, with 30% being removed anaerobically. After 40–50 days of acclimatization the colour removal efficiency reached a maximum, stable value of 90% from a feed dye concentration of 90 mg/l, almost all being removed during the anaerobic phase. This colour removal was attributed to microbial degradation rather than adsorption and colour removal capacity was not lost even after a seven-day absence of dye in the fed substrate. The dye-fed reactor experienced a reduction in the ORP values attained during the non-aerated phase, after acclimatization, an effect not observed in the dye-free control. Under denitrifying conditions it was observed that the decolouration levels achieved in the anaerobic phase decreased from 90% to 70% after only two cycles with a feed containing 45–60 mg NO3/l. Reduction of the SRT value from 15 to 10 days reduced the biomass concentration from 2.0 to 1.2 g VSS/l and lowered colour removal levels from 90% to 30–50%. When the SRT value was increased back to 15 days the colour removal capacity of the system was completely recovered, suggesting that with a SRT of 10 days the adequate microbial population could not be installed in the reactors.

Active chitinases in the apoplastic fluids of healthy white lupin (Lupinus albus L.) plants

Acidic exocellular class III chitinase (EC 3.2.1.14) was previously identified in healthy white lupin (Lupinus albus L.) plants and suspension-cultured cells by N-terminal microse-quencing. In this study, the detection of chitinase activity with Remazol Brilliant Violet 5R (RBV)-labelled chitin derivatives is described. Chitinase activity was observed in protein fractions of cytoplasmic or exocellular origin from roots, hypocotyls, cotyledons, and leaves of healthy white lupin plants. Using isoelectrofocusing followed by a new overlay technique with carboxymethyl chitin-RBV conjugate-containing gel, up to six different chitinase isoforms were visualised. Their activity was distributed fairly evenly within a plant with acidic isoforms predominating in cell walls and basic (or neutral) ones found intracellularly. Exocellular location of some chitinase isoforms were also confirmed by detection of their activities in intercellular washing fluids from white lupin tissues. Chitinase activity was demonstrated in culture filtrates and cell walls of suspension-cultured white lupin cells.

Plasma methylumbelliferyl-tetra-N-acetyl-chitotetraoside hydrolase: further study of its characteristics as a chitinase and comparison with its activity on Remazol Brilliant Violet carboxymethyl chitin

We have further studied some characteristics of human plasma specific chitinase by making use of the fluorescent substrate methylumbelliferyl-tetra-N-acetyl-β- d-chitotetraoside (MU-TACT). The enzyme is also active towards the substrates MU-di-N-acetyl-β- d-chitobioside (MU-DACB) and MU-N-acetyl-chitotrioside (MU-TRACT). MU-TACT hydrolase in plasma is very stable. It is inhibited by the substrate and the product of the reaction and by allosamidin and ethyleneglycolchitin. When the activity of plasma MU-TACT hydrolase was compared to Remazol Brilliant Violet carboxymethyl (RBV) chitin hydrolase (RBV chitinase), it appeared that another enzyme –lysozyme – is also active on RBV chitin and this enzyme represents about 50–60% of the total RBV chitinase activity. Highly increased activity of plasma MU-TACT hydrolase in plasma of Gaucher patients was reflected in a similar increase of RBV chitin hydrolase. In these patients, both MU-TACT hydrolase and RBV chitinase are totally inhibited by allosamidin indicating that specific chitinase is the increased enzyme. With the MU-TACT substrate, specific chitinase is measured and with RBV chitin as substrate the measured activity is a combination of specific chitinase (activity inhibited by allosamidin) as well as lysozyme (residual activity after allosamidin inhibition). For measurement of specific chitinase in human plasma and clinical applications, the di-, tri- or tetra-N-acetylglucosamine derivatives of MU are recommended. In order to avoid confusion, recommended names are either the total substrate followed by -ase, or chitinase.

Dye-labelled substrates for the assay and detection of chitinase and lysozyme activity

A carboxymethyl-substituted soluble chitin and a reprecipitated colloidal chitin derivative were covalently linked with Remazol Brilliant Violet 5R. Both conjugates are new dye-labelled substrates suitable for the assay and detection of chitinase and lysozyme activity. A reliable colorimetric microassay adapted to microtitre plates was established, based on the precipitability of carboxymethyl-chitin-Remazol Brilliant Violet 5R from buffered solutions with hydrochloric acid. Furthermore, sensitive plate-clearing assays were developed for the isolation and detection of chitinolytic microbial populations, and for the screening of pure cultures.

Method for the detection and quantitation of chitinase inhibitors in fermentation broths; isolation and insect life cycle effect of A82516.

A new method of screening for chitinase inhibitors in crude fermentation broths as a means of discovering new insecticidal leads has been developed. In this procedure soluble Remazol brilliant violet 5R dye-coupled chitin degradation products released from insoluble chitin azure substrate by hydrolysis with Streptomyces griseus chitinase are filtered in 0.45 micron Millititer HA 96 well filtration plates and collected in 96 well microtiter plates. Inhibitors of this reaction are detected by a decrease in absorbance (570 nm) of the filtrate. A chitinase inhibitor, designated A82516, produced by culture A82516 was discovered using this screen. Purified A82516 was found to have an IC50 of 3.7 X 10(-6) M for S. griseus chitinase. At a test concentration of 0.27 mg/ml, A82516 was 100% effective in preventing development of house fly larvae to pupae. Allosamidin, a recently reported chitinase inhibitor in vitro, has spectral properties identical to A82516.