An insoluble chromolytic substrate for the determination of proteolytic activity

A new insoluble chromolytic substrate for the determination of proteolytic activity was prepared by immobilization of dyed casein into the structure of polyacrylamide gel. The prepared substrate could detect approximately 0.1 microgram of trypsin per ml. The spontaneous leakage of dyed casein molecules in water solutions was negligible.     

A modified procedure for the detection of microbial producers of extracellular proteolytic enzymes

Summary A simple procedure for the detection of microbial producers of proteolytic enzymes using dyed gelatin microcarrier particles incorporated into appropriate nutrient agar is described. Extracellular proteinases produced by the tested microbial strains hydrolyzed the substrate and clear dyed zones around and under the colonies were formed.     

Determination of the solubilizing activity of a cellulase complex with dyed substrates

When the solubilizing activity of a microbial cellulase complex (e.g.,Trichoderma viride) is determined with conventional methods based on formation of reducing sugars, the results depend on the concentration ratio of cellobiose and glucose in the reaction mixture and thus on the β-glucosidase present and on the type of measurement of reducing sugars. The use of dyed substrates is one way to avoid this problem. The release of coloured compounds from a dyed substrate is proportional to the solubilization.     

Gymnoascus arxii's potential in deteriorating woollen textiles dyed with natural and synthetic dyes

So far, very little is known about microbiological deterioration of dyed woollen textiles. In this paper, the influence of the Gymnoascus arxii fungus on woollen textiles dyed with natural and synthetic dyes was studied. What is more, it was analysed whether the enrichment of the culture medium with additional nutrients has any impact on the deterioration of dyed woollen fabrics caused by a strongly keratinolytic strain. The study was carried out by means of a pure culture method over three different time periods, i.e. 1, 2 and 4 weeks. Within a week, the pure Gymnoascus arxii strain led to a severe deterioration in the mechanical strength of the examined woollen textiles, with the raw fabric being the most severely damaged. After the two-week incubation period, only the fabrics coloured in yellow, i.e. the fabric dyed with natural dye weld, and the synthetic yellow textile as well as the textile dyed with natural dye indigo survived, exclusively on the enriched medium. Solely the weld dyed textile withstood the four-week culture on the nutrient-enriched medium. The conducted studies demonstrated a strong influence of Gymnoascus arxii on dyed fabrics leading to their irreversible destruction. It has been also shown that the presence of nutrients in the substrate that are readily available to microorganism may hinder the development of the Gymnoascus arxii strain and thus, prevent textile deterioration.     

Cellulase Hydrolysis of Cotton Cellulose: The Effects of Mechanical Action,Enzyme Concentration and Dyed Substrates

Experiments with dyed and undyed cotton, using different cellulose concentration and with or with out mechanical action showed a high effect of those factors. The process with mechanical action have a higher weight-loss. Dyed cotton with vat and sulfur dyes have almost die same weight-loss than undyed cotton, but on reactive dyed cotton the cellulase hydrolysis is shorter. It was also verified a decrease of hydrolysis extent with an increase of the reactive dye concentration on the cotton substrate. Some synergism due to the enzyme concentration was apparently observed on the changes of the length of the leaving sugars.     

A continuous spectrophotometric assay for the determination of cellulase solubilizing activity

A cellulase assay was developed for the continuous measurement of colored cellulose oligosaccharides (total carbohydrates) released during enzymatic hydrolysis of dyed crystal-line cellulose. Several cellulosic substrates were uniformly dyed by Remalzol brilliant blue R salt without altering their physical properties. Dyed Avicel (6.5%, w/w) was selected as the most representative substrate for the assay procedure. The assay was performed continuously in a simple, thermally controlled apparatus designed for filtration of the reaction mixture via a 5-μm-pore-size nylon filter to retain the crystalline dyed cellulose while spectrophotometrically monitoring the absorbance at 595 nm of the reaction filtrate. Crude supernatant cellulase of Trichoderma viride QM9414 was used to test the assay procedure. The activity of cellulase on dyed Avicel as measured by ΔA_595nm correlated directly with the total carbohydrates formed. The initial reaction rate of cellulase solubilizing activity was readily determined with high sensitivity. The continuous assay has utility for the study of cellulase kinetics and for the comparison of activities from different microorganisms.     

A dye release assay for determination of lysostaphin activity

We describe a method for determination of lysostaphin activity using Remazol Brilliant Blue R (RBB)-dyed staphylococcal cells or RBB-dyed staphylococcal peptidoglycan as substrate. The dyed substrates are easy to prepare and are stable for at least 6 months. Soluble hydrolytic products released by lysostaphin are measured spectrophotometrically at 595 nm after the insoluble substrate is removed by filtration or centrifugation. The dye release assay is more sensitive and more accurate than the previously described turbidimetric assay.     

Rapid tube test for detecting fungal cellulase production.

A simple procedure is described for the detection of cellulolytic activity in fungi, utilizing dyed cellulose powder as substrate. The powder, incorporated into a basal medium containing agar, mineral salts, and growth factors, is layered onto basal medium minus cellulose. Cellulase production results in release of the dye which diffuses into the lower layer.     

Salinity and temperature effects on accessibility of soluble and cross-linked insoluble xylans to endo-xylanases

Different responses to salinity were observed for an extremely halotolerant endo-xylanase when assayed with soluble birchwood glucoronoxylan and cross-linked dyed insoluble birchwood glucoronoxylan. Shrinking of insoluble xylan particles due to increased ionic strength is proposed as the explanation. Temperature affected the xylanase activity measurement on the insoluble xylan greatly, likely due to increased enzyme accessible surface of the substrate at high temperatures.     

The identification and estimation of elastase in serum and plasma

1. Electrophoretic separation of partially purified elastase preparations from pancreas followed by incubation of the electrophoretogram in contact with an agar gel containing 4% of either Congo Red-stained or unstained elastin demonstrated that the enzyme which dissolves elastin can be identified with that which releases dye from the stained preparation. 2. A method for the estimation of elastase based on the release of dye from Congo Red-stained elastin is described. It is 23 times as sensitive as methods employing protein determination. 3. With the method, elastase activity can be identified in plasma and in a partially fractionated plasma protein preparation. 4. Lineweaver–Burk plots of these estimates of activity at a variety of substrate concentrations indicate that the reaction between elastase and the dyed elastin is more closely similar to that between elastase and the soluble substrate elastin rather than to that between elastase and the solid substrate. 5. Values for K_m and V_max. calculated for the enzyme present in plasma present further evidence for its identity with the pancreatic enzyme. 6. By calculation of the slopes of the Lineweaver–Burk plots for various enzyme concentrations it has proved possible to demonstrate that the inhibitor which is also present in the plasma is without effect on the enzyme when it acts on the dyed substrate.     

Automatic assay of cellulase activity during fermentation

An automatic method using dyed cellulose as a substrate was developed to determine the solubilizing activity of a cellulase complex. The automatic analyzer was connected to a fermentor and enzyme activity was successfully assayed during fermentation. A useful sampling arrangement was developed and the analysis was sensitive enough to make a short reaction time possible. Cellulase production with Trichoderma viride was used as a model process.     

Detection of cellulolytic actinomycetes using cellulose-azure

A rapid method is described for the detection of cellulolytic, thermophilic actinomycetes involving the use of a dyed cellulose substrate (cellulose-azure) in an agar medium. The test was performed in test tubes in which agar containing cellulose-azure was layered on top of a basal medium (mineral salts agar) resulting in two distinct layers. After inoculation, and incubation at 50°C, strains which were cellulolytic caused release substrates for such a test.     

Dyeing of wool fibres with natural dyes: effect of proteolytic enzymes

In spite of the widespread use of proteins (casein, peptone, etc.) and protein fragments as a substrate for the proteolytic enzymes, a substrate prepared from dyes that adsorb onto appropriate materials, such as wool and cotton, are also used for enzyme activity determination. In the point of view of this thought, it was our aim to develop the substrates which are easily and economically obtainable and also environmentally safer for the frequently used proteolytic enzymes, such as subtilisin carlsberg, trypsin, chymotrypsin, and protease type XVI and, if possible, to prepare the specific substrate at least for one of these enzymes. For this aim, wool was dyed with natural dyes such as juglone, lawsone, berberine, and quercetin. The optimum pH, incubation time, and agitation rate were determinated. The results indicate that, of all the tested enzymes on wool-dye complex as an insoluble substrate, the most appropriate complex was found to be wool-lawsone complex.     

Detection of biomolecular interaction between biotin and streptavidin on a self-assembled monolayer using magnetic nanoparticles

For developing a magnetic bioassay system, an investigation to determine the presence of a specific biomolecular interaction between biotin and streptavidin was done using magnetic nanoparticles and a silicon substrate with a self-assembled monolayer. Streptavidin was immobilized on the magnetic particles, and biotin was attached to the monolayer-modified substrate. The reaction of streptavidin-modified magnetic particles on the biotin-modified substrate was clearly observed under an optical microscope. The magnetic signals from the particles were detected using a magnetic force microscope. The results of this study demonstrate that the combination of a monolayer-modified substrate with biomolecule-modified magnetic particles is useful for detecting biomolecular interactions in medical and diagnostic analyses.     

Measurement of α-amylase and glucoamylase activities produced during fermentation

A method for the automatic measurement of α-amylase and glucoamylase activities during fermentation has been developed. Soluble starch dyed with Remazol Brilliant Orange was used as the substrate for α-amylase and 4-nitrophenyl α-d-glucopyranoside for glucoamylase. The same automatic analysis system could be used for both of these enzymes because the reaction products were measured at the same wavelength. Simultaneous pick-up of enzyme and the respective substrate was enabled by using two samplers. The presence of α-amylase did not interfere with the glucoamylase determination. Absolute values for α-amylase activity were obtained using a mathematical correction. Monitoring of these enzymes was accomplished during microbial fermentation.     

Automatic cellulase assay in computer coupled pilot fermentation

Equipment for the automatic measurement of cellulase activity was connected to a computer-coupled pilot fermenter. The measurement was based on the use of dyed Avicel cellulose as substrate. An intermittent sampling device was used to take a sample from the fermenter at intervals of one hour. The computer was programmed to calculate the cellulase activity during fermentation. The measured activity values were corrected against a standard sample of known activity by means of a mathematical model for the calibration curve which was stored in the computer. On-line measurement of enzyme activity was successfully performed.     

Sensitive detection of endo-1,4-beta-glucanases and endo-1,4-beta-xylanases in gels

A simple, highly sensitive zymogram technique for detection of endo-1,4-beta-glucanases and endo-1,4-beta-xylanases in polyacrylamide gels after electrophoresis or isoelectric focusing was developed. The detection employs transparent agar replicas containing soluble covalently dyed polysaccharides, hydroxyethylcellulose dyed with Ostazin brilliant red H-3B and beechwood 4-O-methyl-D-glucurono-D-xylan dyed with Remazol brilliant blue R, as the respective substrates. The high sensitivity of the detection is achieved by selective removal of depolymerized dyed substrates from the agar replicas by solvents which neither solubilize nor precipitate the original nondegraded dyed polysaccharides present in the agar gel.     

Metabolism in marine flatfish--III. Measurement of elastase activity in the digestive tract of Dover sole (Solea solea L.)

A critical assessment of different methods for measuring elastase activity in crude preparations has been made using whole intestinal homogenates of Dover sole. The use of the natural substrate elastin or its dyed derivatives gave optimal pH values in the alkaline region (pH 9.4-9.8) whereas artificial substrates showed optimal hydrolysis nearer neutrality in the region pH 8.1-8.2. Exoproteases may interfere with certain assay procedures. The properties of Dover sole elastase have been further investigated using chromatographic techniques which indicated that the main elastase activity has a molecular weight of approximately 19,500 and an isoelectric point in the region of pH 5.7.     

A novel approach for adding smart functionalities to cellulosic fabrics

A water soluble nanocomposite, based on Ag-nanoparticles (Ag-NPs) loaded on hyperbranched poly (amide-amine, HBPAA) was prepared, characterized and utilized in functional finishing as well as in combined reactive dyeing/and functional finishing of linen, cotton and viscose fabrics. Incorporation of the nanocomposite alone and in combination with reactive dyes in easy care finishing formulations brought about an outstanding antibacterial functionality of the finished and the dyed/finished fabrics even after 15 laundering cycles along with a slight negative impact on other performance properties. Improvement or decrement in the functional, comfort, and dyeing properties is governed by the type of cellulosic substrate.     

Dyed birds achieve higher social status than controls in Harris' sparrows

The status-signalling hypothesis has been proposed to explain colour variation among individuals in flocking birds. Its fundamental assumption is that colour may affect the determination of an individual's social rank in a flock. Here I show for winter Harris' sparrows (Zonotrichia querula) that birds dyed to resemble adults dominate control birds within experimental flocks of young males and young females. The domination of controls by the dyed birds was achieved by a two-step process in both experiments: immediately after the two groups were combined, the controls avoided the dyed birds; then, shortly thereafter, the dyed birds began actively to displace the control birds.