Enzyme filter paper

The following description is from U.S. Patent 2,701,227 To 50 liters of distilled water there was added 10.17 kg of enzyme hydrolyzed casein (N-Z-Amine). The temperature was raised to 100°C and held until the casein digest solution was clear. The container was then cooled rapidly to 15°C and the cooled solution filtered through a coarse grade of filter paper. A small amount of toluene was added as a preservative and the solution... 

Electrolyte leakage. Tissue discs, prepared from potato tubers as described above, were incubated for 16 h at 25°C between wet filter papers. After incubation, the discs were shaken in 20 ml H2O for another 60 min. One ml of this extract was diluted 30-fold with water, and subjected to conductivity measurements using a HI 8788 apparatus (Hanna Instruments). An increase in conductivity indicates a leakage of electrolytes through lesions in the cell wall caused by enzyme action. Control samples were not incubated, they were shaken in water only. 

There are also RMs which are prepared for a specific application and are used for validation of relevant methods. Cobbaert et al. (1999) made use of Ion Selective Electrode (ISE)-protein-based materials when evaluating a procedure which used an electrode with an enzyme-linked biosensor to determine glucose and lactate in blood. Chance et al. (1999) are involved with the diagnosis of inherited disorders in newborn children and they prepared a series of reference materials consisting of blood spotted onto filter paper and dried, from which amino-acids can be eluted and... 

The purple colonies of T. roseopersicina are lifted on a filter paper, transferred onto a stack of filter papers soaked with oxidised redox dye (benzyl viologen) under air. Following heat treatment, the cells containing heat stable, active enzyme turn blue under hydrogen atmosphere,those containing defected hydrogenase remain purple [Fodor et al., 2001],... 

This may be achieved by complete immersion of full-thickness skin in trypsin solution or by placing the heat-separated epidermis for 24 h at 37°C on a filter paper soaked with the enzyme preparation [64, 83]. Other techniques, such as vacuum or chemically induced blistering, stretching, application of staphylococcal exfoliatin, or proteolytic digestion of viable cells, are seldom reported [74, 87-89],... 

The titers of cellulase activities found in anaerobic digesters, when compared to the few other "hydrolytic environments" for which analytical data are available, are strikingly low. Table III shows such values for filter paper and carboxymethyl cellulose degrading activities. This evidence seems to indicate that the cellulose degrading enzymes in ... 

Assays for endo-l,4- -glucanase [EC 3.2.1.4] (i.e., CMCase) and saccharifying cellulase (i.e., international filter paper U, IFPU) activities partially followed the methods recommended in the 1987 lUPAC report (65). When even undiluted enzyme samples fail to give the required glucose yield under prescribed assay conditions, the lUPAC committee recommends a less precise method. In the current study, cellulase activities in digester extracts were so low that the CMCU could only be defined as follows one CMC unit of activity was that amount of enzyme required to liberate one Hg glucose from CMC in 60 min. 

General protease, a-amylase, and exoglucanase activities were estimated using hide powder-, amylose-, and celliilose-azure substrates, respectively, as described earlier (49). Here, standard curves were developed for the hydrolysis of each azure-linked substrate by standard enzymes of known activity. By this method, one cellulose-azure hydrolysis unit corresponds to one filter paper unit, one unit of hide powder-azure activity corresponds to the hydrolysis of 1.0 nmole of iV-benzoyl-L-tyrosine ethyl ester (BTEE) per min, and one amylose-azure unit of activity corresponds to the hydrolysis of 1.0 nmole of maltose from starch per 30 min. 

A great advantage of spectrophotometric assays is that they can be carried out in microtiter plates or as filter paper assays, thus allowing a high sample throughput coupled with small sample volumes. Such systems were used for example for the screening of epoxide hydrolases [34]. A classical example of activity tests is for amylolytic enzymes where starch agar plates are stained with iodine after a certain reaction time. The radius of clear spots is a measure of the reaction rate [35]. 

Figure 5. Reaction mixtures demonstrating short-fiber-forming activities of endo-l,4-/3-D-glucanases. The reaction mixtures are shown after two weeks incubation at 40°C with 20 mg filter paper (3). Represented from left to right are a control without enzyme, Endoglucanase TV (235 fig/ mL), Endoglucanase III (87 fig/mL), and Endoglucanase II (75 fig/mL).

Characterized by release of free fibers from filter paper. Exact role of this enzyme not yet fully understood (5)... 

Coil the filter paper strip and put it in a small test tube. Add 0.5 mL of enzyme solution and 1.0 mL of the buffer solution. Mix on a vortex mixer briefly and incubate for 1 hour at 50°C. Stop the reaction by immersing the test tube into an ice bath. 

The Lobry de Bruyn reaction can be carried out on the paper, followed by chromatography of the products.167 168 The procedure of Bayly and Bourne22 for forming benzylamine derivatives of oligosaccharides on filter paper has been mentioned previously. Enzymic hydrolysis of phosphate esters, in conjunction with paper chromatography, aids detection of the spots.126... 

Filter paper activity, which describes the overall cellulolytic activity of an enzyme preparation, was determined by the method of Mandels et al. (16). A 1 x 6 cm strip of Whatman no.l filter paper (Hillsboro, OR), which equals 50 mg of cellulose, served as the substrate and was added to the sample solution containing 0.5 mL of appropriate diluted enzyme (supernatant of culture broth) and 1.0 mL of 0.05 M citrate buffer (pH 4.8). After 60 min of incubation at 50°C, the hydrolysis was terminated by the addition of 3 mL of DNS solution, and the mixture was further assayed for reducing sugar content by the DNS method. One international filter paper unit (FPU) was defined as the amount of enzyme that releases 1 pmol of glucose/min under the assay conditions. Activities were reported as FPU/milliliter. 

Cellulase activity of the samples was determined as filter paper activity (FPA) expressed in filter paper units (FPU) using Mandels procedure (15), and (3-glucosidase activity was assayed using 4-nitrophenyl-(3-D-glucopyranoside substrate according to Berghem and Petterson s (16) method. All samples were analyzed in triplicate and the mean values were calculated. The relative standard deviation of enzyme activity measurements was always below 5%. 

The purified proteins were assayed for enzyme activity on filter paper, swollen cellulose, BMCC, and CMC following the procedures stated by Irwin et al (5). 

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