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Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Santiago de Compostela, Santiago de Compostela, Spain
1 Corresponding author: mpvilla{at}usc.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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-casein labeled with fluorescein isothiocyanate as substrate. Validation of the modified method was confirmed using natural bacterial, fungal, plant, and animal milk-clotting proteases, as well as a milk-clotting enzyme of recombinant origin. The new modified method described here allowed specific quantification of the activity of milk-clotting proteases in a very sensitive way and permitted determination of the appropriate kinetic parameters of all the enzymes tested, consistent with their origin and degree of purity.
Key Words: milk-clotting protease • enzymatic assay • -casein
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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S1, S2, ß,) that account for nearly 80% of bovine milk proteins (Lucey et al., 2003) and that form soluble aggregates because of the -casein molecules that stabilize the micellar structure. There are several models that account for the special conformation of casein in the micelles (Dalgleish, 1998). One of them proposes that the micellar nucleus is formed by several submicelles, the periphery consisting of microvellosities of -casein (Walstra, 1979; Lucey, 2002). Another model suggests that the nucleus is formed by casein-interlinked fibrils (Holt, 1992). Finally, the most recent model (Horne, 1998) proposes a double link among the caseins for gelling to take place. All 3 models consider micelles as colloidal particles formed by casein aggregates wrapped up in soluble -casein molecules.
The milk-clotting process consists of 3 main phases (Carlson et al., 1987a): 1) enzymatic degradation of -casein, 2) micellar flocculation, and 3) gel formation. Each step follows a different kinetic pattern, the limiting step in milk-clotting being the degradation rate of -casein. The kinetic pattern of the second step of the milk-clotting process is influenced by the cooperative nature of micellar flocculation (Carlson et al., 1987b; Silva and Malcata, 2005) whereas the rheological properties of the gel formed depend on the type of action of the proteases, the type of milk, and the patterns of casein proteolysis (Silva and Malcata, 2005). The overall process is influenced by several different factors, such as pH or temperature (Esteves et al., 2003; Vasbinder et al., 2003).
Milk-clotting proteases act on the soluble portion, casein, thus originating an unstable micellar state that results in clot formation (Vasbinder et al., 2003). Chymosin (EC 3.4.23.4) is an aspartic protease that specifically hydrolyzes the peptide bond in Phe105-Met106 of -casein and is considered to be the most efficient protease for the cheese-making industry (Rao et al., 1998). However, there are milk-clotting proteases able to cleave other peptide bonds in the -casein chain, such as the endothiapepsin produced by Endothia parasitica (Drohse et al., 1989). There are also several milk-clotting proteases that, being able to cleave the Phe105-Met106 bond in the -casein molecule, also cleave other peptide bonds in other caseins, such as those produced by Cynara cardunculus (Lucey, 2002; Esteves et al., 2003; Silva and Malcata, 2005) or even bovine chymosin (Kobayashi, 2004). This allows the manufacture of different cheeses with a variety of rheological and organoleptic properties.
The conventional way of quantifying a given milk-clotting enzyme (Poza et al., 2003) employs milk as the substrate and determines the time elapsed before the appearance of milk clots. However, milk clotting may take place without the participation of enzymes because of variations in physicochemical factors, such as low pH or high temperature (Lucey, 2002; Lucey et al., 2003; Vasbinder et al., 2003). Consequently, this may lead to confusing and irreproducible results, particularly when the enzymes have low activity. At the same time, the classical method is not specific enough, in terms of setting the precise onset of milk gelation, such that the determination of the enzymatic units involved becomes difficult and unclear. Furthermore, although it has been reported that -casein hydrolysis follows typical Michaelis-Menten kinetics (Carlson et al., 1987a), it is difficult to determine with the classic milk-clotting assay.
To overcome this, several alternative methods have been proposed, such as the determination of halo diameter in agar-gelified milk (Poza et al., 2003), colorimetric measurement (Hull, 1947), or determination of the rate of degradation of casein previously labeled with either a radioactive tracer (Christen, 1987) or a fluorochrome compound (Twining, 1984). All these methods use casein as the substrate to quantify proteolytic or milk-clotting activities.
The method proposed in the present paper is the result of a modification to the one described by Twining (1984). The main modification was substituting the substrate previously used (casein) by -casein labeled with the fluorochrome fluorescein isothiocyanate (FITC) to yield the fluorescein thiocarbamoyl (FTC) derivative. This variation allows quantification of the -casein molecules degraded in a more precise and specific way, detecting only those enzymes able to degrade such molecules. The method described by Twining (1984), however, was designed to detect the proteolytic activity of a considerably large variety of enzymes.
Validation of the new modified method described here was carried out using a battery of known milk-clotting proteases of different origin (animal, fungal, microbial, recombinant, or of plant origin) usually used in our laboratory or in the cheese-making industry (i.e., Rhizomucor miehei protease), thus allowing us to study the efficiency of the method. The method proposed here allows the precise characterization of enzymes capable of cleaving -casein. Because -casein degradation leads to the formation of milk clots, here we propose the use of this new method for the detection, quantification, and characterization of enzymes able to originate milk curds useful to the industry for the elaboration of different types of cheese.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Finally, trypsin (Boehringer Ingelheim GmbH, Ingelheim, Germany) was diluted as described by Twining (1984) to a final concentration of 5 µg/mL in 50 mM Tris-HCl, pH 7.5, and 10 mM CaCl2.
FTC--Casein Assay
The assays were carried out as described by Twining (1984) in triplicate with some modifications. One gram of -casein (Sigma-Aldrich, St. Louis, MO) was dissolved in 100 mL of 50 mM carbonate buffer, pH 9.5, containing 150 mM NaCl. Then, 40 mg of FITC was added to the solution, and the mixture was gently stirred for 1 h at room temperature. Free FITC was removed by dialyzing twice against suspensions of 1% charcoal in 2 L of water, then against 50 mM Tris-HCl buffer, pH 8.5 overnight, followed by 50 mM Tris-HCl buffer, pH 7.5. The protein concentration was adjusted to 0.5% with 50 mM Tris-HCl buffer, pH 7.2. The substrate was stored in 1 mL aliquots frozen at –20°C.
The quantity of FITC attached to -casein, 14.12 µg per mg of solid, was determined by comparing this substrate with the commercial one, which was casein-fluorescein isothiocyanate type III containing 61 µg of FITC per mg of solid (Sigma-Aldrich).
The reaction mixture contained 10 µL of the enzymatic sample, 20 µL of 0.01 M potassium phosphate buffer, pH 6.0, and 20 µL of 0.5% FTC--casein. The reaction was carried out in covered 1.5-mL Eppendorf tubes in rotary motion (180 rpm) at 37°C from 15 to 185 min, depending on the assay. The reaction was stopped by adding 120 µL of 5% cold trichloroacetic acid and mixing thoroughly. The tubes were allowed to stand at room temperature (23 to 24°C) for at least 1 h. The TCA-insoluble proteins were sedimented by centrifugation for 5 min at 14,000 rpm. A 60-µL aliquot of the supernatant was diluted with 400 µL of 0.5 M Tris-HCl, pH 8.5.
Aliquots (350 µL) of the samples were placed in 96-well plates (Nunc, Roskilde, Denmark), and the final values were measured using a robotized fluorometer (Luminescence Spectrometer LS50B, Perkin Elmer, Wellesley, MA) with a numeric slit of 2.5 and excitation and emission wavelengths of 490 and 525 nm, respectively.
Appropriate controls were performed by substituting the enzyme with 0.01 M potassium phosphate buffer, pH 6.0. Basal fluorescence levels were subtracted from the resulting values to determine the quantity of free fluorocrome. The values of total protein concentration were determined by Bradford’s method (Bradford, 1976).
A standard plot relating the fluorescence values obtained and the amount of degraded FTC--casein was designed (Figure 1
), taking into account that the FTC measured by fluorescence is released after the -casein proteolysis. This plot was constructed using trypsin for the degradation of increasing quantities of FTC-casein.
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-casein in 1 h at 37°C.
As usual, Michaelis-Menten (Dixon and Webb, 1979) plots were performed as described above but modifying substrate concentrations, which were varied from 0.087 to 1.74 mg/mL. Enzymatic samples from B. licheniformis cultures and from the abomasum of B. arnee bubalis were subjected to inhibition studies using several protease inhibitors reported by Rao et al., (1998); pepstatin A, dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), all from Sigma-Aldrich, and EDTA, from Merck (Darmstadt, Germany). The PMSF, DTT, and pepstatin A were dissolved in 100% ethanol. First, enzymatic samples were incubated for 40 min in the presence of the inhibitors, as described by Croux et al. (1990). These previously treated enzymatic samples were subjected to the reaction protocol described for the FTC--casein assay. The final concentration of the inhibitors in the reaction was 1 mM for DTT, PMSF, and EDTA and 1 µg/mL for pepstatin A. Controls were conducted substituting the inhibitor by ethanol except for the EDTA. Both kinetic and inhibition assays were performed to demonstrate the precision and specificity of the new modified method.
FTC-Casein Assay
This assay was carried out as described by Twining (1984). The substrate used, casein fluorescein isothiocyanate III, was obtained from Sigma-Aldrich. The method consisted of determining the proteolytic activity by measuring the fluorescence values obtained from the free fluorocrome released due to casein degradation. The final fluorescence values were measured using the fluoremeter described above. A standard plot was also constructed following the same strategy as that used for FTC--casein assays (Figure 1
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Comparison Between FTC--Casein and FTC-Casein Assays
A specific enzyme able to act only on -casein (R. miehei protease) and a less specific proteolytic enzyme (trypsin) were tested for comparative purposes. Thus, the enzymatic sample was incubated in the presence of FTC-casein or FTC--casein, and the fluorescence was measured following the FTC-casein assay or the FTC--casein assay, respectively. It is important to note that the concentrations of the enzymatic sample or the different substrates in both methods were the same, although the number of bound FTC molecules per gram of substrate was about 4-fold higher in the case of the commercial substrate (FTC-casein from Sigma-Ald-rich). This variation was taken into account to obtain the final values and for comparative purposes. In fact, in the enzymatic units defined above it was taken into account the relationship between fluorescence and protein values, thus allowing comparison of the activity values by using the linear equation; Y = 3.6949, where X refers to the units of FTC-casein per milliliter and Y refers to the units of FTC--casein per milliliter (R2 =0.9981). Data were compared using statistical tests as described in the following section.
Statistical Analysis
To determine the normal distribution of data, standard kurtosis and standard asymmetry were calculated by measuring the data 20 times. Comparisons of variance were performed using the F-test. Finally, for the design of pair comparison, 1-tailed and 2-tailed Student t-tests were used (Samuels and Witmer, 2003). The Statgraphics Plus software V. 5.1 (StatPoint Inc., Herndon, VA) was used.
Milk-Clotting Assay
These assays were carried out using powdered skimmed milk (Central Lechera Asturiana, Siero, Spain) reconstituted at 26% (wt/vol) in 0.01 M potassium phosphate buffer, pH 6.0, as substrate. The enzyme reactions were carried out at least in triplicate at 37°C in test tubes with 200 µL of enzyme solution and 200 µL of substrate. The milk clots were visualized by turning the tubes upside down.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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-Casein Assay-casein (Figure 1) allowed us to definitively establish a linear relationship between a given amount of fluorescence and the above-defined enzymatic units. In the present work we observed that the value of residual fluorescence showed a linear relationship with the substrate concentration. To standardize the method, here the control values were subtracted from the value of each measurement because negative controls may show variations in values of the different batches. As described by Twining (1984), some FTC release can occur when the samples are thawed. This effect can be avoided by performing the controls as described above. It should be recalled that the experiments carried out by Twining (1984) were done with dilutions of pure enzyme, whereas in the present method the assays were carried out with industrial partially purified enzymes or with enzymes obtained directly from bacterial broths or animal or plant tissues, and, hence, were far from pure.
Validation of the FTC--Casein Assay
To validate the method, samples of known milk-clotting enzymes of different origin (from R. miehei, from recombinant E. coli, from flowers of C. cardunculus, from B. licheniformis, and from the abomasum of B. arnee bubalis) were subjected to the FTC--casein assay designed here.
Linearity of the Method
First, a plot showing the specific activity of 3 different enzymatic samples was drawn (Figure 2), relating the values of the amount of total protein in these samples to the values of the enzymatic units extracted from Figure 1. As expected, the degradation of the labeled substrate showed a linear relationship with the increase in enzyme concentration. The linear relationship observed demonstrates that the new modified method works in a precise fashion. The method allowed precise determination of the enzymatic units and the specific activity values of the enzymes. As shown in Figure 2, the commercial enzyme from R. miehei showed higher specific activity values than the B. licheniformis and B. arnee bubalis enzymes, which behaved as crude enzyme samples. Because of its high specific activity, the commercial enzyme from R. miehei was used to carry out a number of enzymatic assays.
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). The results showed that the linearity claimed in Twining’s method (1984) is applicable to fairly diluted enzymatic samples. Additionally, when the results shown in Figure 3 were further analyzed, it was concluded that it is possible to detect activity in R. miehei samples even when the protein concentration is as low as 2.6 ng of enzyme in the reaction mixture, when the reaction is maintained for 1 h. The FTC--casein method showed its high sensitivity and its stability even when the reactions were carried out over a prolonged time.
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, the activity of the 4 enzymatic samples was measured in the presence of increasing concentrations of substrate. The plots revealed that the kinetic reaction fitted classic Michaelis-Menten kinetics, which is also followed by the first step of milk coagulation: the hydrolysis of -casein (Carlson et al., 1987a). No inhibition effect due to an excess of substrate was detected even at high concentrations of substrate, under the conditions described. The Vmax and Km values of the 4 different enzymatic samples were obtained with the Lineweaver-Burk method (Dixon and Webb, 1979; Figure 5). The method allowed the establishment of the expected values of the kinetic parameters (Table 1).
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. The B. licheniformis milk-clotting protease was inhibited by PMSF, thus indicating the serine proteinase nature of the enzyme. On the contrary, B. arnee bubalis chymosin was inhibited by pepstatin, indicating its aspartic character. The results show that both types of milk-clotting enzymes (serine and aspartic proteinases) can be detected by the modified method reported here because they both produced -caseinolysis, this being the first step of the milk-coagulation process, even though their action on caseins may be different.
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-Casein Assay for Measuring -Casein Degradation-casein assay described here and with a view to determining the specificity of the method for measuring -caseinolytic degradation, we statistically compared this method and the method previously described by Twining (1984). The data obtained are shown in Table 3.
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When variance comparison was designed, the following hypothesis was established:
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As presented in Table 3, in both cases the P-value was more than 0.05, indicating that H0 could not be rejected.
The statistical data obtained from variance comparison indicated that variance values did not differ significantly, which allowed us to establish that both methods are comparables; therefore the 2-tailed and 1-tailed t-tests could be designed.
The 2-tailed t-test was performed following the hypothesis:
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As shown in Table 3, in the case of trypsin data the P-values were greater than 0.05, demonstrating that H0 could not be rejected. In the case of the data for R. miehei, the P-values were less than 0.01, indicating highly significant differences between the means.
The 1-tailed t-test was conducted, establishing the following hypothesis:
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As shown in Table 3, the P-values for trypsin data were greater than 0.05, demonstrating that H0 could not be rejected. Finally, in the case of the data for R. miehei, the P-values were less than 0.01, showing that H1 is acceptable.
The statistical data obtained from the t-tests revealed that the values obtained with the FTC--casein assay are significantly greater (2.648-fold more) than those obtained with the FTC-casein assay when R. miehei protease was used, whereas when trypsin was used the values obtained were similar using both assays or did not differ significantly.
Finally, it was concluded that the method reported here detected -caseinolytic activity more specifically than the one described by Twining (1984). Taking all the above results together, it is possible to state that the new modified FTC--casein method is able to specifically detect -caseinolytic enzymes more efficiently (2.648-fold better) than Twining’s method, whereas the values of the activity of a less specific enzyme (trypsin) were similar using both methods.
Final Comments
At this juncture it is important to remark that all the proteases used in this work produced milk clots when the classic milk-clotting assay was followed (Poza et al., 2003). The classic milk-clotting assay was only qualitative, giving either the actual appearance of milk clots or affording the determination of the most active enzyme of those analyzed. Thus, no comparison between the results obtained from classic milk clotting and FTC--casein assays was possible.
Milk clotting is a complex process that depends on many factors, such as high temperature or low pH (Esteves et al., 2003; Vasbinder et al., 2003). Regardless of these factors, the FTC--casein method affords accurate and precise determinations of -caseinolytic degradation, the first step in the milk-clotting process. The modified FTC--casein assay described here allows the detection of different types of proteases at levels when no milk clotting is yet apparent, unveiling its higher sensitivity over currently used assay procedures. Therefore, the method may find application as an indicator during the purification or characterization of new milk-clotting enzymes.
The method described by Twining (1984) has been extensively used over the last 2 decades to measure the proteolytic activity of all types of protease able to nonspecifically degrade substrates such as casein, hemoglobin, or fibrinogen, allowing the detection of minimum quantities of enzyme; i.e., 5 ng (Twining, 1984). The modified method proposed here basically consisted of the same methodology described by Twining (1984) but employed a more specific substrate and allowed detection of enzyme amount as low as 2.6 ng. The reason for this modification is the need for precise quantification of the activity of the proteases that lead to milk clotting. The use of fluorescence-based measurements allowed precise and fast quantifications of the so-called clotting strength of a given protease. Because variations in the viscosity of milk only take place when the enzymatic phase of coagulation is nearly completed (60% of the elapsed time before clotting appears; Lucey, 2002) and because the time required for the classic assay increases for very diluted enzymatic samples, the FTC--casein assay allows a precise screening of the enzymatic phase during the milk coagulation process. The FTC--casein assay is amenable to use by the cheese-making industry for specifically testing -caseinolytic enzymes of different origin and nature.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Received for publication February 6, 2006. Accepted for publication April 11, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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) and FTC-
). X refers to milligrams of protein per milliliter, and Y refers to fluorescence values. Error bars correspond to standard deviations from triplicate replicas.
). X refers to milligrams of protein per milliliter, and Y refers to activity (U/mL). Error bars correspond to standard deviations from triplicate replicas.
), and Cynara cardunculus (
). Error bars correspond to standard deviations from triplicate replicas.