Cellulase measurement

Loewenberg and Chapman (40), working with a different strain of the same organism, found that there was a 6-hr lag before any CM-cellulase (measured viscosimetrically) could be detected in the extracellular medium, whereas Nisizawa et al. (27) reported a lag period of... 

Viscometric approaches to cellulase measurement activities are important because other methods measure only the number of glyco-sidic bonds cleaved in a polymeric substrate. 

For example, when cell walls of maize stem were treated with sodium hydroxide (0.1M) at 20°C for various times to release different amounts of phenolics, a highly significant correlation (r = 0.98) was found between the amount of phenolics released and wall biodegradability (measured by cellulase ) (5). It is of interest to note that alkali treatment of poor quality graminaceous forages (e.g., cereal straw) is used commercially to increase their biodegradability, and thus their feed value for the animal (1). 

SAXS measurements with CBH II indicated a very similar tertiary structure for both CBH I and CBH II, in spite of a different domain arrangement (to be published). Discrete differences in the tail parts could, however, be noticed. The maximum diameter of CBH II (21.5 nm) was higher than in CBH I this might be due to duplication of the glycosylated part in the former case. Thus, the functional differentiation of these cellulases can be reflected by structural differences. 

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 ... 

The pretreatment of wastewater with hydrolases or acids is one of the best ways to overcome this obstacle, because in this way the big polymer molecules can be decomposed to smaller units, which can be measured by the biosensor. The positive effect of an enzymatic pretreatment of wastewater prior to sensorBOD measurement was demonstrated [53, 66]. In these investigations, different types of wastewaters, which contained milk powder, starch, or cellulose, were treated by proteases, a-amylases, and cellulases or a mix of these enzymes, respectively. This pretreatment resulted in a good correlation between sensorBOD and the conventional five-day BOD, while the sensorBOD values for untreated wastewater were significantly lower (see Table 6). As an example, the sensorBOD of a wastewater from a paper factory increased approximately to the fourfold value when treated by a mixture of cellulase and -glucosidase. 

There are two reasons for the measurable cellobiose concentration in the T. viride cellulase hydrolyzed syrups. The most likely is that T. viride has rather poor / -glucosidase activity so that cellobiose accumulates. Evidence of this is that additions of / -glucosidase to the T. viride cellulase improves its activity. A second reason is that the / -glucosidase enzyme is strongly glucose inhibited. Hence the rate of cellobiose hydrolysis slows down as the glucose concentration rises, allowing cellobiose to accumulate. 

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. 

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. 

A third water-soluble substrate for endocellulase is hydroxyethyl-cellulose (HEC). As early as 1931, Ziese (5) used HEC and Sandegren et al. (6) defined the activity as being proportional to the change of the inverse of the specific viscosity per time unit. Child et al. (7) have also used HEC and based their calculation -on a linearization of the viscosity measurements according to Eriksson et al. (8). They defined an enzymic activity unit as the amount of enzyme causing a viscosity change of 0.001 rjre 126 min"1. The exponential factor was used in order to linearize the data. This unit is useful for comparison of cellulases of different origin, but it is based on an empirical relationship. The authors made an evaluation of their method in comparison with CMC hydrolysis. 

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. 

We thus elucidated that three of the four cellulase components are endo- or random-type and the other is exo-type. However, it is difficult to distinguish between the components of least or lowest random-type and those of exo-type. It is rather easy to identify an endo-type cellulase component. In contrast, it is very difficult to determine a cellulase to be exo-type because if the enzyme has a glycosyl-transferring activity the hydrolysis product is not a single sort, which is one of the necessary conditions to be an exo-type. Based on our experiments, measurement of the time course of CMC using a sample of medium substitution degree seems to be the best method of diagnosis to determine a cellulase component to be endo- or exo-type. With some enzymes, direction of mutarotation of reaction products is useful to resolve this problem, as is illustrated by the classic example of the starch hydrolysis by a- and /3-amylases. If this is true for our cellulases, the mutarotation of reaction products would be a... 

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 ... 

Dissolve 0.1 g of cellulase in 10 mL of 0.1 M NaAc buffer. You are going to measure the enzyme activity of this solution. 

Mandels, M., R. Andreotti, and C. Roche, "Measurement of Saccharifying Cellulase,"Biotechnol. Bioeng. Symp. 6 (1976) 21-33. 

The objective of the current work was to characterize carefully the dynamics of cellulase production and metabolic activity following cellulose addition in a batch cultivation of the strain T. reesei Rut-C30. Cells were initially grown on glucose as the carbon source, and after its depletion, cellulose was added. Since it is difficult to follow the growth directly after addition of a solid substrate, on-line measurements of C02 evolution were used to follow the metabolic activity of the cells. Frequent samples were also taken to measure enzyme activity and sugar concentrations. 

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