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Article

Alcalase Specificity by Different Substrate Proteins Under Different Conditions: The Enzyme Immobilization on Carrageenan Beads Strongly Affects the pH/Activity Curve Depending on the Substrate Protein

by
Alan Portal D’Almeida
1,2,
Pedro Abellanas-Perez
1,
Luciana Rocha Barros Gonçalves
2,
Tiago Lima de Albuquerque
3,
Ivanildo José da Silva Junior
2,* and
Roberto Fernandez-Lafuente
1,*
1
Department of Biocatalysis, ICP-CSIC, Campus CSIC-UAM, 28049 Madrid, Spain
2
Department of Chemical Engineering, Federal University of Ceará, Pici Campus, Fortaleza 60420-275, Brazil
3
Department of Food Engineering, Federal University of Ceará, Pici Campus, Fortaleza 60420-275, Brazil
*
Authors to whom correspondence should be addressed.
Catalysts 2025, 15(8), 750; https://doi.org/10.3390/catal15080750
Submission received: 4 July 2025 / Revised: 31 July 2025 / Accepted: 4 August 2025 / Published: 5 August 2025
(This article belongs to the Section Biocatalysis)
Browse Figures
Versions Notes

Abstract

Alcalase was immobilized–stabilized on carrageenan beads following a previously described protocol. Then, the activities of free and immobilized enzymes were compared using different protein substrates (casein, (CS), bovine serum albumin (BSA), or hemoglobin (HG)) at different pH values and temperatures. The observed activity depended on the substrate protein and enzyme formulation used. The highest enzyme activity could be observed at pHs 5, 7, or 10, depending on the substrate protein and the Alcalase formulation. The effect of the temperature at these pHs on the activity versus the different substrate proteins showed a common pattern. At low temperatures, the immobilized enzyme presented higher (mainly at acidic-neutral pH values and using BSA) or similar specific activity than the free enzyme. At temperatures near the optimal for the free enzyme, it became the most active, while at higher temperatures, the immobilized enzyme recovered the lead, although differences in the optimal temperature were not very significant. This may be explained by the lower mobility of the immobilized–stabilized enzyme. The immobilized enzyme could be much more active than the free enzyme or slightly less active, even using mild conditions, depending on the substrate protein, pH, and temperature used to determine the enzyme activity.

1. Introduction

Interest in enzyme biocatalysis is steadily growing due to the good properties of these biocatalysts, such as high activity at moderate temperatures and pressure, high selectivity and specificity, which permit performing the reaction under environmentally friendly conditions, and preventing the formation of by-products [1,2,3,4]. In this context, proteases have a high interest [5,6,7,8]. The main commercial use of proteases is in detergent formulations [9,10,11], but they are also used to produce bioactive peptides through the hydrolysis of proteins from different sources [12,13,14,15,16,17,18,19], to catalyze amidation reactions to produce peptides [20,21,22,23,24,25,26,27,28], to resolve racemic mixtures, [29,30,31,32,33,34,35], etc. While biocatalysis should compete in the production of chemicals with conventional organic chemistry, the enzymes (cell-free or inside the microorganisms) have no rival in the modification of foods due to the importance of these processes of the absence of undesired by-products.
As stated above, one of the main applications of proteases is the hydrolysis of proteins [12,13,14,15,16,17,18,19]. It may be expected that a specific protease can recognize different peptide bonds in the substrate protein, and that the catalytic efficiency of a protease versus a specific protein may depend on the exposition of these bonds, the environment generated by the other groups in the protein substrate, etc. In addition, it may be expected that, if some fragments of the substrate protein are released due to proteolysis, the exposition of some groups of the substrate protein can change (and even of the fragments), becoming more or less accessible to the proteolysis [36]. This diversity of reactions simultaneously occurring implies that the concept of a Michaelis constant is implausible, and so we will be measuring simultaneous, multiple reactions, each with its own Km and Kcat. Moreover, it becomes more complex when considering that the protease can also be a substrate, and this will be more relevant when the concentration of the substrate protein is reduced. Thus, while for small and monofunctional substrates (e.g., esters of amino acids), the Km and Kcat of a protease may have real meaning, considering the hydrolysis of the protein, this can be more complex, and its meaning may not be realistic. Moreover, it may be expected that different substrate proteins can provide different reaction rates as a result of the number of peptide bonds susceptible to being hydrolyzed by the protease, their exposition to the medium, etc. In addition, this may differ if the experimental conditions change. Now, the activity is not only the result of a more efficient active center of the protease but also the exposition of the peptide bonds that can be hydrolyzed by the enzyme [36].
Even though enzymes have many features that are positive for their use as industrial biocatalysts, their biological origins pose some features that are inadequate for this use [37]. As a result, in many instances, many enzyme features need to be improved; for example, enzymes should be utilized for long time periods at the industrial level. Therefore, the stability of an industrial enzyme needs to be high enough under operational conditions to be useful. Enzyme specificity and selectivity in the physiological environment and with the natural substrate need to be exhibited under very different conditions and with substrates different from the physiological ones. Enzyme inhibition, necessary in vivo, is only a problem in industry since regulation is not required, delaying the reaction or even preventing it from reaching the target yield. Fortunately, nowadays, there are many tools to obtain enzymes suitable for industrial applications. Metagenomics allows the utilization of full biodiversity (including non-cultivable organisms or even no-longer-existing ones) [38,39,40,41]; directed evolution permits improves in specific enzyme features in the desired direction by mimicking natural evolution in an accelerated way [42,43,44]; modelling and directed mutagenesis allow for the generation of new active centers (converting inert proteins into enzymes or enzymes into so-called plurizymes) [45,46,47,48]. After investments are made to design the desired enzyme, these tools enable the user to obtain an improved enzyme produced at a price similar to the native enzyme. These enzymes may be subject to additional physicochemical modifications to further improve the enzyme features, but this time, changes need to be performed each time a new batch of biocatalysts is prepared. In this sense, the chemical modification of enzymes is becoming more site-directed by the day, enabling the improvement of results [49,50,51,52]. Enzyme immobilization is a last resort to improve enzyme features, compatible with all previous techniques, which can be utilized to improve enzyme immobilization and not to improve the intrinsic properties of the enzyme [53,54,55]. This tool was initially developed to solve the problem of enzyme recovery and reuse [56,57,58]; nowadays, it has proved to be able to improve many other enzyme features: stability, activity, selectivity, specificity, inhibitions, etc., and may be improved if the immobilization protocol is properly performed [59,60,61,62,63,64].
The immobilization of proteases has some specific problems and advantages [5,63,65,66,67]. As a main advantage, immobilization of a protease inside a porous support (but not in a non-porous nanoparticle [68]) avoids enzyme autolysis [63]. This not only has a positive effect on enzyme stability but also prevents protease peptides from being incorporated into the final product. The main problem is that if the immobilized protease is used to hydrolyze proteins, enzyme orientation regarding the support surface can be a key point for the final protease activity, even if the active center remains intact [68].
Enzyme immobilization may alter the enzyme structure and increase its stability; as such, some changes in the immobilized protease specificity for different proteins can be expected, as well as perhaps a different response to the change in reaction conditions. This has scarcely been studied, but it may have certain relevance [36]. Recently, our research group has presented the optimization of the immobilization of Alcalase in carrageenan beads [69]. Alcalase is an alkaline serine endopeptidase initially produced by Bacillus subtilis, commercialized by Novozymes, that is produced by submerged fed-batch fermentation using Bacillus licheniformis, and it is also called “Subtilisin Carlsberg”, subtilisin A, or subtilopeptidase A [70]. This formulation was initially designed for its use in detergents [71], but it may be utilized in bating and the dehairing of leather, improving the digestibility of animal feed, helping in cheese flavor development, producing meat tenderizing, the production of bioactive peptides [17,72,73,74], etc. Alcalase prefers to hydrolyze large uncharged residues in the acyl donor position, but it can hydrolyze many other groups of proteins [75,76]. This enzyme has been immobilized following different protocols, leading to improved properties [77,78,79,80,81,82,83,84]. In this paper, a recently reported protocol has been utilized to immobilize Alcalase: the optimal immobilization of Alcalase in carrageenan beads permitted to enhance its stability [69].
In this new research effort, the activities of free and immobilized Alcalase have been compared at different pH values using bovine serum albumin (BSA), casein (CS), and hemoglobin (HG). The isoelectric points of the different enzyme preparations are 4.6–4.7 for casein, 6.8 for HG, and 4.9 for BSA. Furthermore, in some selected pH values, the effect of temperature on the activity of these enzyme formulations was evaluated. From our understanding, this is the first analysis on how the joint effect of the proteolysis pH value and enzyme immobilization can tune the activity of proteases versus different protein substrates, and how this can respond to changes in the experimental conditions.

2. Results and Discussion

2.1. Immobilization of Alcalase and Preliminary Evaluation of the Enzyme Specificity Versus Different Substrate Proteins

Figure 1 shows the immobilization course of Alcalase in carrageenan beads following the optimal conditions previously described [69]. Immobilization proceeded quite rapidly. Table 1 shows the activities of free and immobilized Alcalase versus the different proteins at pH 8. The free enzyme has more activity versus HG, and the lowest activity is versus CS (about 60%). The immobilized enzyme has almost the same activity as the free enzyme using CS, a 12% higher activity using BSA, and a 10% lower activity using HG. Therefore, the immobilized enzyme presented similar activities with BSA and HG, while CS was the substrate that presented the lowest activity. These results already show how the observed activity of both Alcalase formulations depends on the substrate protein and how the immobilization of the enzyme produced changes in the enzyme specificity.

2.2. Effect of the pH Value on the Activity of Different Alcalase Formulations Versus Different Proteins

First, we would like to remark that Alcalase activity could not be found in the supernatant after incubating the biocatalyst in the pH range from 4 to 10 at 25 °C after 2 h. Therefore, we can assume that the release of the enzyme to the reaction medium cannot explain the observed results.
Moreover, adsorption of the substrate protein in the matrix cannot be discarded; in fact, it very likely occurred, mainly at acidic pH values. However, the amount of substrate protein, 10 mg/mL, means that there was 100 mg of substrate protein per g of support. This largely exceeded the adsorption capacity of the support [69], but some protein adsorption cannot be discarded on the support, mainly using pH values where the support and the proteins had an ionically opposite nature. This could have reduced the available substrate protein for the immobilized protease, thus promoting an apparent decrease in the enzyme activity. However, as it can be observed in the obtained results discussed below, this seems not to have had a significant negative effect on the activity of the immobilized enzyme.
Figure 2, Figure 3 and Figure 4 show the effect of the pH on the activity of both Alcalase formulations with the three studied protein substrates. We have described relative activity considering 100% the activity/mg of the enzyme at the pH value where the maximum activity value is achieved using the free or the immobilized enzyme. It is remarkable that the activity/pH curves are very different for each substrate protein, also changing with the utilized Alcalase formulation.
Using CS (Figure 2), the free enzyme activity increased steadily from pH 5 (at pH 4, we were unable to dissolve the casein at the target concentration) to pH 10, decreasing at pH 11. With this protein, the immobilized Alcalase exhibited a higher activity than the free enzyme at pHs 5–7 (almost doubling the activity of the free enzyme at pH 5), while there was slightly lower activity from pH 8 to pH 11. The activity of this immobilized biocatalyst increased from pH 5 to pH 7, decreased at pH 8, while at pH 9, it was mostly unchanged; it increased again at pH 10, decreasing later at pH 11. While the free enzyme had a clear maximum activity at pH 10, the immobilized enzyme had two activity-optimal pH values: one at pH 7 and the other at pH 10.
The results are very different using BSA (Figure 3). The free enzyme had a broad pH range, where the activity was very similar, from pH 6 to pH 8, with the activity at pH 5 being very similar to these values. Then, at higher pH values, the Alcalase activity decreased progressively. In this instance, at pH 10 (optimal value in the hydrolysis of CS), the activity was clearly lower than the activity in the flat region. The situation was very different when using the immobilized enzyme. Here, the highest activity was found at pH 5, and then the activity slowly decreased as the pH increased. At pHs 4–6, the activity of the immobilized enzyme was higher than that of the free enzyme (at pHs 4 and 5, it almost doubled the activity of the free enzyme), while at pHs 9–11, the most active biocatalyst was the free enzyme (although by a narrow margin). Although BSA has some tendency to aggregate, at acidic pH values [85,86,87,88,89,90,91,92], the structure and oligomerization of BSA should be identical using free or immobilized enzymes, so the differences should be based on the different enzyme conformations of the free and immobilized enzymes.
HG is a multimeric enzyme; therefore, the possibility of subunit dissociation may have some influence on the results [93,94,95] (dissociation is favored at alkaline pH values [96]). Using this substrate protein (Figure 4), the free enzyme activity increased when the pH value increased from 4 to 7, and then decreased progressively until pH 11. In this instance, the differences between free and immobilized enzyme activity/pH curves were not very relevant (just a slightly higher activity at an acidic pH for the immobilized enzyme and a higher activity at an alkaline pH for the free enzyme). In any case, the optimal pH for both Alcalase preparations was pH 7.
Therefore, not only did the substrate protein hydrolyze with different efficiencies depending on the enzyme formulation [36], but the substrate protein also altered the “optimal” pH for the activity of the different enzyme formulations, which, in some instances, were very different from each other.

2.3. Effect of the Temperature on the Different Alcalase Formulations at Different pH Values

We have analyzed the effect of temperature on both Alcalase formulations at pHs 5, 7, and 10, which were found to be the optimal values for enzyme activity at 25 °C for some biocatalysts and some substrate proteins in the previous section.
Figure 5 shows the results using CS at the different pH values. At pH 5, the activity of the free enzyme was lower than that of the immobilized enzyme until it reached 35 °C; when performing the reaction at 40 °C, the free enzyme experienced a large increase in activity, surpassing the activity of the immobilized enzyme. The free enzyme reached a maximum at 45 °C (remaining more active than the immobilized enzyme), and then decreased, similar to the immobilized enzyme. However, the decrease in activity of the immobilized enzyme was slower than that observed using the free enzyme; at 50 °C, its activity became higher than that of the free enzyme again, and at 55 °C, it almost doubled that of the free enzyme. Nevertheless, the free enzyme had more activity than the immobilized enzyme at the optimal temperature. At pH 7 (Figure 5), the situation was similar: the immobilized enzyme was more active than the free enzyme at temperatures from 4 to 35 °C; then, the free enzyme became more active than the immobilized enzyme until it reached the maximal activity (in this instance, at 50 °C). For both enzyme formulations, the highest activity was detected at this temperature. At 55 °C, the immobilized enzyme retained 50% more activity than the free enzyme. At pH 10, the situation followed a similar pattern but with different values (Figure 5). Here, at 4 °C, the immobilized enzyme was slightly more active than the free enzyme, which showed higher activity in the range 25–45 °C, where the free enzyme had the highest activity under these conditions and substrate. The immobilized enzyme presented the highest activity at 50 °C, surpassing the activity of the free enzyme under these conditions, and retained more than 50% more activity than the free enzyme at 55 °C. The maximum activity with this substrate (which we have called 100%) was always obtained using the free enzyme at 45 °C. However, in many instances, at lower and higher temperatures, the immobilized enzyme was more active. The results suggest that at certain temperatures (different at each pH value), the Alcalase-free enzyme suffered a change in its conformation, which produced a more active enzyme and may not have been observable using the immobilized enzyme, perhaps because the enzyme support-interactions increased the enzyme rigidity and reduced the possibilities of these changes. At higher temperatures, these changes in the Alcalase conformation made this enzyme less efficient, and again, this occurred with more difficulty using the immobilized enzyme, which, despite usually maintaining the same optimal temperature as the free enzyme (except at pH 10, where the enzyme is less stable and the immobilization increases this optimal temperature), retained much more activity at higher temperatures.
The situation was not very different using BSA, but there were some differences (Figure 6). At pH 5, the immobilized enzyme was much more active than the free enzyme, mainly at 25 °C; at 4 °C, the differences were smaller. Both enzyme formulations had optimal activity at 40 °C (using CS was at 45 °C). At 40 and 45 °C, the free enzyme was slightly more active than the immobilized enzyme, while the immobilized enzyme was more active at 55 °C. At pH 7, the immobilized enzyme was more active than the free enzyme from 4 to 35 °C, while the free enzyme was more active at 40 and 45 °C. Optimal activity for both Alcalase formulations was achieved at 45 °C. The immobilized enzyme recovered the lead in activity at 50 and 55 °C (at 55 °C, it was almost 50% more active). At pH 10, the free enzyme was slightly more active than the immobilized one from 4 to 45 °C (optimal temperature for the free enzyme). The immobilized enzyme had the highest activity at 50 °C, only surpassing the activity of the free enzyme at 50 °C and 55 °C (Figure 6). The differences in response to the temperature changes between the free and immobilized enzymes might have been related to differences in the mobility of the enzyme, as explained above. The small differences in the results using BSA and CS were likely caused by differences in the substrate protein (the formation of aggregates and conformational changes that exposed hidden peptide bonds susceptible to being attacked by the enzyme).
Finally, the same study was performed using HG (Figure 7). In this instance, at higher temperatures, a higher protein subunit dissociation was expected. At pH 5, the immobilized enzyme was more active than the free enzyme from 4 to 35 °C, while the free enzyme was slightly more active at 40–42.5 °C. The optimal activity was observed at 42.5 °C for the free enzyme and at 45 °C for the immobilized biocatalyst. The immobilized enzyme was again the most active at 45 °C, tripling the activity of the free enzyme at 55 °C. The picture was very similar at pH 7 (optimal activity for the free enzyme was 45 °C and 47.5 °C for the immobilized enzyme); the free enzyme only surpassed the activity of the immobilized enzyme at 40–45 °C. At pH 10, the free enzyme was slightly more active than the immobilized enzyme from 4 to 40 °C (optimal temperature for the free enzyme); the activates of both biocatalysts were very similar at 42.5 °C (optimal activity for the immobilized enzyme), while at higher temperatures, the immobilized enzyme became more active (with around 40% more activity at 55 °C).
In all cases, the highest activity was obtained using the free enzyme, although the immobilized enzyme presented similar or higher optimal temperatures. However, the free enzyme was less active than the immobilized enzyme at low temperatures at pH 5 and 7, and at temperatures above the optimal one. At pH 10, the free enzyme was more active than the immobilized one at low temperatures in many cases. The optimal temperature was similar or higher for the immobilized enzyme, but the retention of activity at higher temperatures was higher for the immobilized enzyme. For this enzyme, the optimal temperature ranged between 40 and 45 °C using the free enzyme with all substrate proteins and pH values, while the immobilized enzyme had this optimal value at higher temperatures in some instances. The change in the substrate protein also promotes some changes in the activity/temperature profile, but the changes were smaller than the pH value.

3. Materials and Methods

3.1. Materials

Novozymes (Madrid, Spain) were kindly provided by Alcalase®Pure from B. licheniformis (EC 3.4.21.62) in a liquid formulation. Ethylenediaminetetraacetic acid (EDTA), hemoglobin from bovine blood (HG), and L-cysteine were obtained from Sigma-Aldrich (Madrid, Spain). Thermo-Fisher (Alcobendas, Spain) supplied calcium chloride. Tokyo Chemical Industry Europe (Zwijndrecht, Belgium) supplied trichloroacetic acid (TCA). The carrageenan applied in the research was extracted from Solieria filiformis seaweed cultivated in Trairi-CE, Brazil, which was gently furnished by the Algae Producers Association from Flecheiras and Guajiru. The method described by Bradford [97] was employed to determine the protein concentration. All other reagents were of analytical grade.

3.2. Methods

3.2.1. Preparation of Carrageenan Beads

Solieria filiformis seaweed was used to obtain ι-carrageenan by alkaline hydrolysis and ethanol extraction, as described in a previous report [69]. A 20 mL solution of 1% (w/v) ι-carrageenan was prepared using distilled water and heating it up to 50 °C until it was dissolved. Next, samples of 10 µL of solution were slowly dripped into a 100 mL solution of 5% (w/v) CaCl2, pH 7.0, at 25 °C. The beads were left under constant agitation for two hours. Finally, they were filtered, washed with excess distilled water, and stored at 4 °C. This protocol yielded beads of around 1 mm in diameter (too large or small beads were manually discarded).

3.2.2. Enzyme Immobilization

The Alcalase immobilization was performed, as previously described [69], by mixing 2.0 g of carrageenan with 40 mL of 200 mM of Tris-HCl solution pH 8.0 (adjusted with 1.0 M HCl and 1.0 M NaOH), and then adding 66 µL of an enzyme commercial solution (~33 mg/mL), giving a support enzyme load of 1.0 mg Alcalase/g of support. Then, the system was incubated at 25 °C for 2 h. The immobilization was monitored by measuring the protease activity of the supernatant and a free enzyme reference solution under identical conditions. The produced biocatalyst was recovered by filtration and rinsed with an excess of distilled water [69]. All enzyme activity was immobilized on the support [69].

3.2.3. Activity of the Different Alcalase Formulations in the Hydrolysis of BSA, CS, and HB

The hydrolysis of BSA, CS, and HB was performed, as described by Kunitz [98]. First, for the evaluation of the effect of the pH on the enzyme activity at 25 °C, 100 mM buffers were produced at different pHs: sodium acetate at pHs 4.0 and 5.0, sodium phosphate at pHs 6.0 and 7.0, and sodium carbonate buffer at pHs 8–11.0. Then, 1% (w/w) BSA, CS, or HB solutions were prepared using different buffers and a combination of 5 mM EDTA and 5 mM cysteine. To initialize the reaction, 30.3 µL of a solution of 3.3 mg/mL of free Alcalase or 100 mg of immobilized biocatalyst (the same amount of protein in both cases) was added to 10 mL of the substrate solution and incubated at 25 °C for 15 min under gentle agitation. Then, samples of 1.0 mL were withdrawn and added to 2.0 mL of a 10% TCA solution to cease the reaction and force the aggregation of non-hydrolyzed proteins, leaving as the only soluble protein fraction that which proceeded from the released peptides. The reaction suspension was centrifuged at 12.500 rpm for 2 min, and the increment in the absorbance of the supernatant containing released peptides was evaluated at 280 nm. As a reference, the intact protein substrate solution was submitted to the same treatment. A protease activity unit was defined as a 0.001 increase in absorption per minute−1 under the defined conditions.
At some selected pH values, the temperature was altered in the range 4–55 °C to determine the effect of the temperature on the hydrolytic activity of the free and immobilized Alcalase versus the different proteins. Thus, the selected buffers containing the substrates were incubated in shakers at the desired temperature (for 1 h to reach the target temperature); then, the biocatalyst was added to the reaction media, and then the activity evaluation was performed, as described above, adding 10% TCA and determining the absorbance at 280 nm of the supernatant [98]. An ice bath was utilized to keep the temperature at 4 °C.

4. Conclusions

The results presented in this paper show that the substrate protein not only affected the observed activity of Alcalase but also demonstrated how the activity of the enzyme responded to changes in the reaction conditions. The effect of the pH fully depended on the used substrate; maximum activity was found at pH 10 using CS, but it was at pH 7 using HG, while it was flat between pHs 5 and 7 using BSA. We have reported this for the first time. Curiously, these differences are not so large when analyzing the effect of temperature on the enzyme activity (at different pH values) on the activity of free Alcalase; the curves were not identical, but were not very different when changing the protein substrate. The immobilization of the enzyme in carrageenan by cation exchange, which produced some stabilization of the enzyme, changed this picture. The effect of the enzyme immobilization on the specificity of a protease at different pH values and temperatures has not been reported in any previous paper. This immobilized–stabilized enzyme, with a lower mobility, presented similar (mainly at pH 10) or significantly more activity (at pH 5 and 7) than the free enzyme at low-moderate temperatures (depending on the substrate protein and analyzed pH value); at temperatures near the optimal ones for the free enzyme, this enzyme formulation took the lead, and at temperatures above this value, the immobilized enzyme returned to be the most active. Therefore, depending on the pH and the temperature, immobilization produced enzyme hyperactivation or a small decrease in the enzyme activity, even if the activity was determined under mild conditions. In fact, for BSA, a maximum activity might have been obtained using the immobilized enzyme at pH 5. This can be explained by the fact that the free enzyme suffered a drastic increase in enzyme activity at specific temperatures (different at each pH value), perhaps due to a conformational change, which was not observed using the immobilized enzyme. The used substrate protein was also relevant for these changes in enzyme activity due to alterations on the reaction conditions.
Therefore, the results presented in this paper show that it is not easy to describe enzyme specificity. One substrate protein may be preferred under one set of specific reaction conditions, while another can be preferred under different conditions. The picture has been shown to be completely different if an immobilized enzyme is used.

Author Contributions

Conceptualization, R.F.-L. and I.J.d.S.J.; formal analysis, T.L.d.A., A.P.D., P.A.-P. and R.F.-L.; investigation, A.P.D. and P.A.-P.; data curation, A.P.D., P.A.-P. and R.F.-L.; writing—original draft preparation, All authors; writing—review and editing, All authors; supervision: R.F.-L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), by a granted scholarship and funding (Process nº PS1-0186-00043.01.00/21); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Grant nº. 440639/2022-0/2022-9).

Data Availability Statement

The data will be available upon request.

Acknowledgments

The authors would also like to thank the Algae Producers Association of Flecheiras and Guajiru cities for the algae supply, the Grupo de Pesquisa em Termofluidodinâmica Aplicada (GPTA), and Central Analítica UFC/CT-INFRA/MCTI-SISANO/Pró-Equipamentos CAPES for the support. RFL wants to thank the support from Ministerio de Ciencia e Innovación and Agencia Estatal de Investigación (Spanish Government) (PID2022-136535OB-I00). The help and suggestions of Angel Berenguer (Universidad de Alicante) during the writing of this paper are gratefully recognized.

Conflicts of Interest

The authors declare no conflicts of interest.

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Catalysts 15 00750 g001
Figure 1. Immobilization course of Alcalase in 1.0 mm carrageenan beads. The experiment was carried out using 200 mM TRIS/Cl at pH 8.0 and 25 °C. Further specifications can be found in Section 3.2. Circles: free enzyme; Triangles: supernatant.
Figure 1. Immobilization course of Alcalase in 1.0 mm carrageenan beads. The experiment was carried out using 200 mM TRIS/Cl at pH 8.0 and 25 °C. Further specifications can be found in Section 3.2. Circles: free enzyme; Triangles: supernatant.
Catalysts 15 00750 g001
Catalysts 15 00750 g002
Figure 2. Effect of the pH on the activity of Alcalase versus CS at 25 °C. Experiments were performed for 15 min. Activity is expressed in percentage of activity (%) relative to the most active enzyme, free or immobilized. Further specifications can be found in Section 3.2. Circles: free enzyme; Triangles: immobilized enzyme.
Figure 2. Effect of the pH on the activity of Alcalase versus CS at 25 °C. Experiments were performed for 15 min. Activity is expressed in percentage of activity (%) relative to the most active enzyme, free or immobilized. Further specifications can be found in Section 3.2. Circles: free enzyme; Triangles: immobilized enzyme.
Catalysts 15 00750 g002
Catalysts 15 00750 g003
Figure 3. Effect of the pH on the activity of Alcalase versus BSA at 25 °C. Experiments were performed for 15 min. Activity is expressed in percentage of activity (%) relative to the most active enzyme, free or immobilized. Further specifications can be found in Section 3.2. Circles: free enzyme; Triangles: immobilized enzyme.
Figure 3. Effect of the pH on the activity of Alcalase versus BSA at 25 °C. Experiments were performed for 15 min. Activity is expressed in percentage of activity (%) relative to the most active enzyme, free or immobilized. Further specifications can be found in Section 3.2. Circles: free enzyme; Triangles: immobilized enzyme.
Catalysts 15 00750 g003
Catalysts 15 00750 g004
Figure 4. Effect of the pH on the activity of Alcalase versus HG at 25 °C. Experiments were performed for 15 min. Activity is expressed in percentage of activity (%) relative to the most active enzyme, free or immobilized. Further specifications can be found in Section 3.2. Circles: free enzyme; Triangles: immobilized enzyme.
Figure 4. Effect of the pH on the activity of Alcalase versus HG at 25 °C. Experiments were performed for 15 min. Activity is expressed in percentage of activity (%) relative to the most active enzyme, free or immobilized. Further specifications can be found in Section 3.2. Circles: free enzyme; Triangles: immobilized enzyme.
Catalysts 15 00750 g004
Catalysts 15 00750 g005
Figure 5. Effect of reaction temperature in the activity of Alcalase versus CS at pH 5.0 (A); pH 7.0 (B) or pH 10.0 (C). Hydrolyses were performed for 15 min. Activity is expressed in percentage of activity (%) relative to the most active enzyme, free or immobilized. Further specifications can be found in Section 3.2. Circles: free enzyme; Triangles: immobilized enzyme.
Figure 5. Effect of reaction temperature in the activity of Alcalase versus CS at pH 5.0 (A); pH 7.0 (B) or pH 10.0 (C). Hydrolyses were performed for 15 min. Activity is expressed in percentage of activity (%) relative to the most active enzyme, free or immobilized. Further specifications can be found in Section 3.2. Circles: free enzyme; Triangles: immobilized enzyme.
Catalysts 15 00750 g005
Catalysts 15 00750 g006
Figure 6. Effect of reaction temperature in the activity of Alcalase versus BSA at pH 5.0 (A); pH 7.0 (B) or pH 10.0 (C). Hydrolyses were performed for 15 min. Activity is expressed in percentage of activity (%) relative to the most active enzyme, free or immobilized. Further specifications can be found in Section 3.2. Circles: free enzyme; Triangles: immobilized enzyme.
Figure 6. Effect of reaction temperature in the activity of Alcalase versus BSA at pH 5.0 (A); pH 7.0 (B) or pH 10.0 (C). Hydrolyses were performed for 15 min. Activity is expressed in percentage of activity (%) relative to the most active enzyme, free or immobilized. Further specifications can be found in Section 3.2. Circles: free enzyme; Triangles: immobilized enzyme.
Catalysts 15 00750 g006
Catalysts 15 00750 g007
Figure 7. Effect of reaction temperature in the activity of Alcalase versus HG at pH 5.0 (A); pH 7.0 (B) or pH 10.0 (C). Hydrolyses were performed for 15 min. Activity is expressed in percentage of activity (%) relative to the most active enzyme, free or immobilized. Further specifications can be found in Section 3.2. Circles: free enzyme; Triangles: immobilized enzyme.
Figure 7. Effect of reaction temperature in the activity of Alcalase versus HG at pH 5.0 (A); pH 7.0 (B) or pH 10.0 (C). Hydrolyses were performed for 15 min. Activity is expressed in percentage of activity (%) relative to the most active enzyme, free or immobilized. Further specifications can be found in Section 3.2. Circles: free enzyme; Triangles: immobilized enzyme.
Catalysts 15 00750 g007
Table 1. Activity of free and immobilized Alcalase at pH 8 and 25 °C versus different proteins. The activity is given, as described in Section 3.2. (0.001 increment of Absorbance per mg and min). Other specifications may be found in Section 3.2.
Table 1. Activity of free and immobilized Alcalase at pH 8 and 25 °C versus different proteins. The activity is given, as described in Section 3.2. (0.001 increment of Absorbance per mg and min). Other specifications may be found in Section 3.2.
Alcalase FormulationSubstrate Protein
CSBSAHG
Free3.14 ± 0.184.12 ± 0.115.26 ± 0.22
Immobilized2.98 ± 0.134.64 ± 0.214.77 ± 0.12
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MDPI and ACS Style

D’Almeida, A.P.; Abellanas-Perez, P.; Gonçalves, L.R.B.; de Albuquerque, T.L.; da Silva Junior, I.J.; Fernandez-Lafuente, R. Alcalase Specificity by Different Substrate Proteins Under Different Conditions: The Enzyme Immobilization on Carrageenan Beads Strongly Affects the pH/Activity Curve Depending on the Substrate Protein. Catalysts 2025, 15, 750. https://doi.org/10.3390/catal15080750

AMA Style

D’Almeida AP, Abellanas-Perez P, Gonçalves LRB, de Albuquerque TL, da Silva Junior IJ, Fernandez-Lafuente R. Alcalase Specificity by Different Substrate Proteins Under Different Conditions: The Enzyme Immobilization on Carrageenan Beads Strongly Affects the pH/Activity Curve Depending on the Substrate Protein. Catalysts. 2025; 15(8):750. https://doi.org/10.3390/catal15080750

Chicago/Turabian Style

D’Almeida, Alan Portal, Pedro Abellanas-Perez, Luciana Rocha Barros Gonçalves, Tiago Lima de Albuquerque, Ivanildo José da Silva Junior, and Roberto Fernandez-Lafuente. 2025. "Alcalase Specificity by Different Substrate Proteins Under Different Conditions: The Enzyme Immobilization on Carrageenan Beads Strongly Affects the pH/Activity Curve Depending on the Substrate Protein" Catalysts 15, no. 8: 750. https://doi.org/10.3390/catal15080750

APA Style

D’Almeida, A. P., Abellanas-Perez, P., Gonçalves, L. R. B., de Albuquerque, T. L., da Silva Junior, I. J., & Fernandez-Lafuente, R. (2025). Alcalase Specificity by Different Substrate Proteins Under Different Conditions: The Enzyme Immobilization on Carrageenan Beads Strongly Affects the pH/Activity Curve Depending on the Substrate Protein. Catalysts, 15(8), 750. https://doi.org/10.3390/catal15080750

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