Enzyme Assays Cellulase Activity

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. 

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. 

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... 

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... 

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. 

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%. 

Enzymes were immobilized onto silica gel by covalent crosslinking with glutaraldehyde in a procedure similar to that of Kondo et al. (3). Briefly, 1-4 g of silica gel was incubated in 1-13 wt% glutaraldehyde and in 100-1000 mL of enzyme solution for up to 48 h. The immobilized enzyme was recovered by filtration and washed to remove loosely bound enzyme. Samples of the soluble enzyme were collected before and after immobilization and assayed for activity, to provide an estimate of enzyme uptake onto the support. The a-amylases studied were Spezyme Fred (Genencor), Allyzme (Alltech), and Liquozyme (Novozymes). The cellulases studied were Spezyme CP and Spezyme CE (Genencor). 

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. 

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%. 

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-... 

Figure 6. pH-activity profile of cellulolytic enzyme activities from Thermoactinomyces sp. under assay conditions. (Q) CM-cellulase, incubation time 10 min (A) Avicelase, incubation time 20 min (O) /3-glu-cosidase, incubation time 30 min. 

The possibility that BS and BI cellulase could act synergistically, as has been recorded for many components of fungal cellulolytic complexes (1,2), was tested in several assay systems by adding the enzymes separately or together at the same total activity levels (CMCase units). The assays included the hydrolysis of CMC, cellohexaose, and cellulose powder. The results (not shown here) indicated that the pea cellulases were no more or less effective when added together than when added singly, i.e., there is no indication of any interaction between the enzymes, or any preference by one for the products generated by the other. 

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. 

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