The first event in an enzymatic protein hydrolysis process is the addition of enzyme to the protein solution. To compare hydrolyses, the systems should be standardized to a certain extent. For enzymatic hydrolyses performed with different enzymes, the standardization is typically based on the activity (g x U/g) of the enzyme preparation, rather than on the total weight, since the enzyme preparations used may contain other proteins, or carbohydrates or salts, next to the enzyme. The enzyme activity is generally defined as the rate at which a substrate is consumed or the rate at which products are formed during enzymatic incubation. One enzyme unit U is defined as the amount of enzyme which will catalyze the conversion of one μmole of substrate per minute under given conditions. While this definition seems simple, the fact that for proteases different activity assays with different types of substrate are available indicates that the use of the unit is not unambiguous.
Protease activity assays are generally based on colorimetric evaluation of product formation or substrate consumption, using either synthetic peptides, or (labeled) proteins. The azocasein assay, for instance, uses the hydrolysis of a labeled protein. The activity is calculated from the release of peptides containing the label in the solution after 10 minutes at a defined pH and temperature of hydrolysis, after precipitating the intact protein. While the activity determined using this assay is valid for this specific substrate, it may not be the same for other substrates. Between substrates a number of parameters can change, such as the number of bonds that can be potentially cleaved and the accessibility to these bonds. The latter depends on the conformation of the protein. In the same way, to compare the activity of two enzymes, the choice of the substrate is important. For two enzymes with different specificities, the number of potential bonds to cleave on the model substrate will be different. Hence this makes it difficult to find one substrate to compare two enzymes with different specificities.
To illustrate the difficulties in defining the protease activity, additional data collected during the project are presented here. The activity of two enzymes with the same specificity was determined by the azocasein assay. (table 1) The two enzymes used were BLP (Bacillus licheniformis protease, (NS-37005)), obtained from Novozymes (Bagsvaerd, Denmark) and the enzyme V8 from Staphylococcus aureus (#70213122) obtained from Roche (Almere, The Netherlands). Both enzymes are specific for Glu and Asp residues and have been used in previous studies for the hydrolysis of whey proteins [1,2].
The activity as determined by the azocasein assay was expressed in increase of absorbance unit (AU) per time and per mg enzyme [3]. The activity were found to be 0.26, and 3.0 AU/mgenz/min for BLP and V8, respectively. In addition to the standard assay, the hydrolysis of azocasein was also monitored using the pH-stat method. In contrast to the results of the standard assay, the activity determined from the initial slope of the degree of hydrolysis (DH) versus time curves, was similar for both enzymes: 0.02 s-1. (table 1)
Table 1. Activity of BLP and V8 towards azocasein determined by azocasein assay and with the pH-stat method and for β-lactoglobulin determined by the pH-stat method.
The activity determined by the standard azocasein assay actually shows the loss of intact protein. Consequently, the comparison of the two enzymes with this assay is as much a comparison of the mechanism of hydrolysis as a comparison of the activity. Since for both enzymes the initial part of the DH versus time curve (obtained by the pH-stat method) was similar, this shows that V8 has a higher activity towards intact azocasein than BLP.
Since the enzyme activity may also depend on the substrate, both enzymes were also tested on β-lactoglobulin as a substrate in the pH-stat. On this substrate, BLP has a higher activity than V8 as determined by the initial rate on the DH curves. (table 1) The activity determined from the initial rate is again different from the results determined with azocasein as substrate. This confirms results obtained in a previous study showing a higher rate of hydrolysis on β-lactoglobulin with BLP than for V8 using the pH-stat [4]. (figure 1A) In that experiment, similar activities of enzyme, based on azocasein assay were used to hydrolyze the β-lactoglobulin. This study also showed that V8 had a lower affinity towards intact β-lactoglobulin than BLP. (figure 1B)
Figure 1. (A) Hydrolysis curves and (B) Proportion of remaining intact β-lactoglobulin as a function of the degree of hydrolysis for hydrolysis of 0.5 % (w/v) β-lactoglobulin by BLP and by V8. Adapted from [4].
These experiments show that depending on the analysis method and substrate, BLP had either a higher, an equal, or a lower activity than V8. A similar observation has been made for two a-specific protease preparations both containing Subtilisin as the main component, that were compared in two assays [5]. In the azocasein assay, Alcalase 2.4 had a higher activity than Prolyve 1000 while on another substrate (succinyl-Ala-Ala-Phe-paranitroanilide) Alcalase had a lower activity than Prolyve. The activity values, determined by azocasein, were used to standardize the hydrolysis of
Substrate Azocasein β-lg
Enzyme BLP V8 BLP V8
Assay (AU/mgenz/min) 0.26 3.0
pH-stat initial rate (s-1) 0.02 0.02 0.01 0.0003
0 1 2 3 4 5 6 7 8 0 500 1000 1500 2000 2500 3000 DH ( % )
Hydrolysis time (sec)
A BLP V8 100 80 B BLP V8 0 In ta ct β- la ct ogl ob ul in Degree of hydrolysis (%) 0 3 6 9
WPC. Still, a higher DH was reached for Alcalase (19 %) than for Prolyve (15 %) after 5 hours of hydrolysis. This shows that the reaction cannot be standardized based on the activity determined by azocasein assay.
These results show that while the activity assay may be useful to guarantee reproducibility (if the same assay is used under the same conditions), it does not guarantee ‘the same’ activity for enzymes on different substrates. Hence, it is important to develop methods to provide an insight on the molecular mechanism of the enzyme action. This can only be done by characterizing the protein hydrolysates and in particular the kinetics of formation of single peptides