Measurement of saccharifying cellulase
© Eveleigh et al; licensee BioMed Central Ltd. 2009
Received: 19 June 2009
Accepted: 01 September 2009
Published: 01 September 2009
This article sets forth a simple cellulase assay procedure. Cellulose is variable in nature, insoluble and resistant to enzymatic attack. As a result there have been a bevy of bewildering cellulase assays published that yielded irrational results. Certain protocols focused on the rapidity of the assay while ignoring that only the most readily susceptible cellulose regions were being hydrolyzed. Other assays simplified the system by using modified soluble substrates and yielded results that bore no relationship to the real world hydrolysis of insoluble cellulose. In this study Mandels, Andreotti and Roche utilized a common substrate, Whatman filter paper. Hydrolysis of a 50 mg sample of the paper was followed to roughly 4% degradation, which circumvented the problems of attack of only the most susceptible zones. This common hydrolysis target range also resulted in some balance with regard to the interaction of the several cellulase components. The method was subsequently widely adopted.
Douglas E Eveleigh
p-Nitro β glucosidase
Endo β 1, 4 glucanase
Loss in viscosity
Exo β 1, 4 Glucanase
C1 + Cx
Loss in weight
Reduction in optical density (OD)
Uptake of alkali
Filter paper cellulase
Release of dye
Cellulase is a complex of enzymes containing chiefly endo and exo β glucanases plus cellobiase. For complete hydrolysis of insoluble cellulose, synergistic action between the components is required. Since different cellulase preparations vary widely in the proportions of the different components, depending on source, growing conditions of the organism, and harvesting and handling procedures, the rate and extent of their hydrolysis of cellulose substrates also varies widely. Assay of purified components requires a variety of fairly complicated procedures that can be confusing to persons whose chief interest is in practical applications. In Finland (1975) the question arose 'What one substrate can be used to measure all the cellulase components?' Dr L G Petterson  opted for cellotetraose because it is acted on by all known members of the cellulase complex. Dr G Halliwell  decided on cotton because only a complete cellulase will hydrolyze it. So we had the choice of the most susceptible substrate or the most resistant, but both are unsatisfactory for a practical assay. Cellotetraose is not available commercially, but would have to be prepared by the investigator, a major research effort in its own right. Cotton is so slowly hydrolyzed that meaningful assays require 24 h. Finally, neither cotton nor cellotetraose is representative of a realistic substrate.
Filter Paper (FP) Activity and Unitsa
ml enzyme for 2.0 mg glucose
Effect of Conditions on Filter Paper Assaya
Filer paper mg
Enzyme protein mg
Activity mg glucose
Activity per h
It was obviously time to start using a cellulase unit based on the international unit system but two problems arose. The first problem was what concentration of cellulose to use in the assay and the second problem was what extent of conversion was required for meaningful results. For a soluble substrate the answers are simple. Substrate level should be high enough that it does not limit the reaction, and the extent of conversion should be slight before depletion of the substrate or product inhibition affects the reaction rate. Because of the low bulk density of cellulose, concentrations greater than about 5% become very thick and since cellulose is insoluble, the effective concentration is the available surface. Increasing the effective cellulose concentration by adding more of it or by milling the cellulose will increase the rate of the reaction and also make it more linear to a higher sugar value  but this increases the relative contribution of the enzymes acting on the more amorphous portions of the cellulose. We are more interested in the hydrolysis of the more crystalline and resistant portions by the whole cellulase complex. Filter paper, like other insoluble celluloses is a multiple substrate ranging from free ends and amorphous regions to crystalline fibers.
Moving to a more resistant substrate makes for a more rigorous assay but even cotton contains a little amorphous cellulose. For example, Stutzenberger  reported that the cellulase of Thermomonospora curvata contained C1 based on reducing sugar production from cotton. The international unit values looked pretty good because the assay time was only 10 min but the sugar level did not increase even after 30 h incubation. So the action appears to be on a limited (less than 1%) amorphous portion of the cotton. When we use cotton as a substrate, we incubate for 24 h and expect 5% to 10% conversion by Trichoderma preparations. Another proposal by Naylor  was to run the filter paper hydrolysis for a longer time and use the slope of the hydrolysis curve after 16 h. This is scientifically sound but time consuming and tedious if large numbers of assays are to be run.
Our solution to the problem has been to stay with the 50 mg of filter paper and use 0.5 ml of enzyme with 1 h incubation and to calculate international units as shown in Figures 3 and 4 from the dilution to give 2.0 mg of glucose (0.37 units/ml if the 0.5 ml assay is used). This cut-off value of 2.0 was chosen because the hydrolysis curves are fairly linear to beyond that level and because it represents 4% hydrolysis of the filter paper, well over the amount of sugar that could be expected from an incomplete cellulase. Higher unit values would result if the cutoff value were lower, if the assay time were decreased, or the cellulose concentration were increased.
In conclusion, the measurement of cellulase is complex and there is no absolute unit as can be measured for a single enzyme acting on a soluble substrate. The unit value will depend on the substrate chosen, its concentration, and the extent of conversion. The filter paper assay and unit value described here is not perfect but it is simple, reproducible, and quantitative and predicts enzyme action under practical saccharification conditions.
This article originally appeared in Biotechnol Bioeng Symp 1976, 6:21-33. Republished with permission of John Wiley & Sons, Inc.
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