Assay of cellulase activity

A wide variety of substrates have been used for the assay of cellulase activity. 

For the assay of cellulase activity it is desirable to use a method by which the number of cellulase units can be directly determined. A number of quantities are defined in terms of units of enzyme, such as the concentration of an enzyme, normally given as units/ml., the specific activity defined as units/mg. of protein, and the molecular activity, defined as units//unole of enzyme. The determination of units of activity does not present any difficulties for enzymes where the change of the substrate can be expressed in absolute terms. However, for enzymes whose activity is not measured in terms of a chemical reaction but in terms of some physical change, such as a decrease in viscosity of the substrate, complications arise and it has not been possible to express the activity in the units mentioned above. 

Two types of organisms, Gram-positive cocci and Garm-negative rods predominate in the cellulose enrichment cultures. Assay of cellulase activity after four days of incubation indicates that by ultrasonic vibration of organisms attached to cellulosic fibers as much as seven times the amount of activity in the supernatant could be obtained (Table IX). 

Assay of Endocellulase Activity. Cellulase is an enzyme complex a synergistic action between the components is required for a complete hydrolysis of the insoluble cellulose. There is no consensus about the substrate to be used for the cellulase activity measurements. 

The categories of substrates which are used for assays of cellulase enzymes are shown in Table I. The use of crystalline, insoluble forms of cellulose as substrates makes assays difficult and has led to such trivial names as Avicelase activity. These assays are useful as indications of the capacity of an enzyme system to degrade native cellulose and indicate the presence of CBH enzyme which cannot be assayed in the presence of endoglucanases or / -glucosidase. The susceptibility to enzymatic attack generally increases with the hydration of the polymer chains that accom-... 

Cell walls, total membrane-bound components, and ribosomes were separated and assayed for cellulase activity to study the subcellular localization of the enzymes as follows. Segments (approx. 5 g fresh wt) were ground in two volumes of extraction medium containing 0.4M sucrose (ribonuclease-free), 5mM Mg acetate, lOmM Tris-HCl (pH 7.5 at 22°C), 20mM KC1 and 5mM / -mercaptoethanol. The brei was filtered and the filtrate centrifuged at 500 Xg for 20 min. The post-500 Xg supernatant was fractionated essentially as previously described (28). Aliquots (7 mL) of the supernatant were layered on a discontinuous gradient composed of 2 mL 70% (w/v) sucrose and 3 mL 15% (w/v) sucrose both in lOmM Tris-HCl (pH 7.5 at 22°C), lOmM KC1, 2.5mM Mg acetate and ImM / -mercaptoethanol. The tubes were centrifuged at... 

Lists on assay methods of cellulase activity are given in most of the review literature concerning cellulose degradation (14, 26, 34y 41, 47). 

Under the conditions of the assay employed, cellulase activity could not be detected in the supernatant fluid (enzyme fraction 3) of rumen contents. Enzyme fraction 2 which was obtained by ultrasonic disruption of cells in the liquid portion of rumen contents exhibited appreciable activity on CMC but not on Avicel or Solka Floe. On the other hand,... 

A new procedure described for the determination of cellulase activity is based on incubation of the enzyme with finely divided cellulose at pH 6.9 and determination of the D-glucose and cellobiose liberated as their trimethylsilyl derivatives by g.l.c. The method, although generally applicable, was specifically developed for measurement of cellulase activity in mixed enzyme preparations from sheep rumen contents the coefficient of variation of the assay was 2.4-4.5%. 

New sensitive assays for cellulase activity use as substrates modified carboxy-methylcelluloses containing either 2,4,6-trinitrophenyl groups (for spectrophoto-metric assay) or fluorescamine groups (for fluorometric assay). A simple and rapid staining procedure for qualitative and quantitative determinations of plant Citrus sinensis) and fungal T. viride) cellulases is based on the incorporation of carboxymethylcellulose into polyacrylamide gels. After electrophoresis of the crude extract, the gel is incubated in a phosphate buffer, acidified, and treated with iodide-iodine no colour develops in areas containing cellulase activity. 

By utilizing commercially available antibiotic filter discs, which are slightly more resistant to cellulase activity and have a higher degree of crystallinity than conventional filter paper, an improved assay for cellulase activity has been developed. ... 

Carboxymethylcellulose has been used as a solid substrate for the qualitative measurement of cellulase activity of micro-organisms. The substrate allows more rapid assay than does native cellulose. 

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. 

Overall crude cellulase activity was measured using Remazol brilliant blue acid-swollen cellulose (cellulose-azure, Calbiochem.) (22). The assay consists of combining 40 mg cellulose azure, 1.0 mL of lOOmM sodium citrate buffer (pH 4.8), and 1.0 mL of enzyme solution and... 

Table II. Summary of Various Cellulase Activity Assays Incu- bation Volume Time Temp Product ...

Inhibition Studies. A number of compounds were employed to study the amino acid residue(s) that are important for cellulase activity. Samples of enzyme (0.1 mL, 500 units) were pre-incubated with 0.1 mL of inhibitor in semimicroviscometers for 8 min at 35°C. CM-cellulose solution (0.8%, w/v), which had been separately equilibrated at 35°C for 20 min was added to the viscometers and initial viscosity losses were measured after 15 min. Inhibitors were replaced by buffer in control experiments. Compounds that are insoluble in buffer, e.g., N-ethylmalei-mide, diisopropyl fluorophosphate, and succinic anhydride, were dissolved in a small volume of 95% ethanol before assay. p-Chloromercuribenzoate (p-CMB) was first dissolved in 0.2M NaOH and the pH adjusted to eight prior to pre-incubation with cellulases. 

Figure 3. Development of BS and BI cellulase activity in apices of pea seedlings. Intact seedlings were sprayed with the auxin analogue 2,4-D and decapitated seedlings were painted with the natural auxin IAA with or without an inhibitor of DNA synthesis, FUdR. All treatments resulted in massive swelling at the pea apex because of cell expansion cell divisions also occurred, but not in the presence of FUdR (6). Cellulases were extracted as described in Figure 1 and assayed in unpurified form.

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%. 

Each cellulase class has had its share of assay development. Assays that specifically measure each class are difficult to establish, as crossreactivity between classes is greater than zero. However, there are assays that can be used to determine the relative ratios of these activities. 

Esterases. Acetyl esterase (EC 3.1.1.6) removes acetyl esters from acetylated xylose and short-chain xylo-oligomers. It s polymeracting counterpart, acetyl xylan esterase (EC 3.1.1.72), has a similar activity, but prefers polymeric xylan.244 In addition to acetate-specific enzyme detection kits, HPLC or GC analysis of acetate release from native extracted xylan and chemically acetylated xylan, colorimetric substrates, such as p-nitrophenol acetate and /3-napthyl acetate, or the fluorometric substrate, 4-methylumbelliferyl acetate are also used to assay acetyl esterases.244,253 The third esterase, ferulic acid esterase (EC 3.1.1.73), hydrolyzes the ester bond between ferulic acid or coumaric acid and the arabinose side chain of arabinoxylan. Assays for this activity are usually carried out using starch-free wheat bran or cellulase-treated gramineous biomass as a substrate and monitoring ferulic or coumaric acid released by HPLC or TLC. When preparing enzyme-treated substrates, care must be taken to employ phenolic-acid-esterase-free cellulases.244 Other substrates include methyl and ethyl esters of the phenolic acids, as well as finely ground plant biomass.240,254,255... 

Regardless of the assay used, the non-hnearity of cellulase kinetics requires that the enzyme activity be measured based on a fixed level of conversion. 

CN 06897, and Rohm Tech Inc., Malden, MA 02148. All preparations were assayed for polygalacturonase (PG), cellulase, and pectinesterase (PE) activities by the methods of Vas, et al. (6 ) as modified by Bruemmer et al. ( ] ). PG and cellulase activities were determined by reduction of specific viscosity in an Ostwald-type viscosimeter held in a 50°C water bath. PG activity was measured on both 1.5% polygalacturonic acid (U.S. Biochemical Corp.), titrated... 

The disadvantages, then, of the high molecular weight substrates in study of the mode of action of cellulases are many and derive from inherent properties of the substrate molecules themselves. But in spite of the obvious disadvantages, modified celluloses have been useful in simple assays for determining cellulase activity. Recently, fresh attempts... 

For the last few years, we have investigated the cellulase components of Pseudomonas fluorescens var. cellulosa in special regard to the physiological relation of their synthesis and localization to the cultural conditions. This pseudomonad is an aerobic mesophilic cellulose-decomposing bacterium isolated earlier by Ueda et al. (39) from field soil. Some of the enzymatic properties of cellulases obtained from it have previously been reported (28). In the present review, the results of our recent studies (42) are described and discussed together with the related works of other authors. [In these studies, activities of cellulase and aryl / -glucosidase were assayed by the same methods as described in a recent paper (31), and that of amylase by the blue value method modified by Fuwa (10)]. 

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