Cellulase activity measurement

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. 

In 1987, after significant effort, an international committee of cellulase researchers and the International Union of Pure and Applied Chemists (IUPAC) produced a procedure seeking to standardize cellulase activity measurements. This procedure uses microcrystalline cellulose and the dinitrosalicylic (DNS) acid method of Miller186 to measure reducing... 

Chromophoric substrates were also used as tools in the study of the binding of several cellulase components to their natural substrates (such as Avicel). This is illustrated here in the investigation of the synergy in binding of CBH I and CBH II from Trichoderma reesei onto Avicel. The enzymes were differentiated with CNPL (see above), which was a substrate only for CBH I (core I). Thus, the amount of CBH II adsorbed when a mixture of both enzymes was added, either simultaneously or sequentionally, to Avicel was calculated from the amount of CBH I bound (activity measurements with CNPL) subtracted from the values for total protein binding (280 nm absorbance reading). The results obtained from these experiments are summarized as follows ... 

A limitation of the use of ionic substrates is due to the fact that viscosity measurements depend then on ionic strength, pH, and polyvalent cations, making the comparisons of cellulase activities at different pH values difficult. It is generally necessary to dialyze each enzyme preparation or to use a heavily buffered substrate. 

The E-3 peak was high in Avicelase activity and in protein content as compared with CMCase activity. This peak was further fractionated on a Bio-gel P-100 column five protein peaks (E-3-1 to E-3-5) were obtained, of which E-3-2 peak was highest among them in Avicelase activity and protein content. The elution patterns are shown in Figure 3, and the time course of hydrolysis of CMC by these cellulase fractions measured by a decrease in the viscosity is shown in Figure 4. Randomness of them is in the order of E-3-5 < E-3-2 < E-3-1 E-3-4 E-3-3. The E-3-2 fraction was subjected to further purification on a CM-Sephadex C-50 column because E-3-5 was very low in the Avicelase activity. 

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... 

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. 

Frequently we use filter paper as a substrate for the measurement of cellulase activity because it is well defined and yields reproducible results. The activity (IU) we obtain with filter paper is referred to as filter paper unit (FPU). The experimental procedure to measure the FPU of cellulase (Mandels et al., 1976) is as follows ... 

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%. 

Ghose, T. K., Measurement of cellulase activities. Pure Applied Chemistry 1987, 59 (2), 257-268. 

Johnston, D. B., Shoemaker, S. P., Smith, G. M., and JR, W., Kinetic measurements of cellulase activity on insoluble substrates using disodium 2,2 bicinchoninate. J Food Biochem 1998,22, 301-319. 

The determination of glucose is one of the most frequently perfomed routine analyses in clinical chemistry as well as in the microbiological and food industries. Here, the application of glucose electrodes appears to be the method of choice. Moreover, in combination with other enzymes, glucose oxidase sensors are applicable to the measurement of di- and polysaccharides and amylase and cellulase activity, which is required in many biotechnological processes. This versatility explains, why numerous researchers worldwide are concerned with the development and optimization of glucose sensors. 

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... 

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. 

In order to avoid the complications arising from the use of CMC samples of too low and too high DS values, samples in the range 0.8 < DS < 1.0 were recommended for comparative studies of cellulase activity. To make comparisons of absolute activity determinations meaningful it was necessary to determine the numerical values of the quotient c and the Staudinger exponent x for the CMC samples studied. With CMC samples in the mentioned DS range the following conclusions can be drawn from measurements with four different cellulases ... 

Filter paper degrading cellulase activity was measured by the author s (39) shaking method using 2 sheets of Toyo filter paper No. 2, 10 mm. X 10 mm. at 40°C., pH 5.0. Complete degradation was shown by + + ++. 

Cellulase activities were determined on filtrates of culture samples by production of reducing sugar measured as glucose by a dinitrosalicylic acid (DNS) method (32) as follows ... 

Cellulase activity was measured by reducing sugar released from filter paper (Whatman No.l) and from carboxymethylcellulose with glucose as the standard [20-22]. 

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%. 

Activity measurement of classical cellulases can be performed by measuring reducing sugars e.g. BCA or DNS), but for this oxidizing enzyme such methods cannot be used, as was reported by Harris et al. However, they can he used to reflect the synergetic action of the PMO together with the classical cellulases. 

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