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Bromelains are cysteine peptidases with endopeptidase action (a subfamily of papains), obtained from different parts of vegetable belonging to the Bromeliaceae family. They have some intrinsic medical activity, but this review is focused on their application (individually or mixed with other proteases) to produce bioactive peptides. When compared to other proteases, perhaps due to the fact that they are commercialized as an extract containing several proteases, the hydrolysates produced by this enzyme tends to have higher bioactivities than other common proteases. The peptides and the intensity of their final properties depend on the substrate protein and reaction conditions, being the degree of hydrolysis a determining parameter (but not always positive or negative). The produced peptides may have diverse activities such as antioxidant, antitumoral, antihypertensive or antimicrobial ones, among others or they may be utilized to improve the organoleptic properties of foods and feeds. Evolution of the use of this enzyme in this application is proposed to be based on a more intense direct application of Bromeliaceae extract, without the cost associated to enzyme purification, and the use of immobilized biocatalysts of the enzyme by simplifying the enzyme recovery and reuse, and also making the sequential hydrolysis using diverse proteases possible.

Pepsin is a protease used in many different applications, and in many instances, it is utilized in an immobilized form to prevent contamination of the reaction product. This enzyme has two peculiarities that make its immobilization complex. The first one is related to the poor presence of primary amino groups on its surface (just one Lys and the terminal amino group). The second one is its poor stability at alkaline pH values. Both features make the immobilization of this enzyme to be considered a complicated goal, as most of the immobilization protocols utilize primary amino groups for immobilization. This review presents some of the attempts to get immobilized pepsin biocatalyst and their applications. The high density of anionic groups (Asp and Glu) make the anion exchange of the enzyme simpler, but this makes many of the strategies utilized to immobilize the enzyme (e.g., amino-glutaraldehyde supports) more related to a mixed ion exchange/hydrophobic adsorption than to real covalent immobilization. Finally, we propose some possibilities that can permit not only the covalent immobilization of this enzyme, but also their stabilization via multipoint covalent attachment.

Pepsin is a protease used in many different applications, and in many instances, it is utilized in an immobilized form to prevent contamination of the reaction product. This enzyme has two peculiarities that make its immobilization complex. The first one is related to the poor presence of primary amino groups on its surface (just one Lys and the terminal amino group). The second one is its poor stability at alkaline pH values. Both features make the immobilization of this enzyme to be considered a complicated goal, as most of the immobilization protocols utilize primary amino groups for immobilization. This review presents some of the attempts to get immobilized pepsin biocatalyst and their applications. The high density of anionic groups (Asp and Glu) make the anion exchange of the enzyme simpler, but this makes many of the strategies utilized to immobilize the enzyme (e.g., amino-glutaraldehyde supports) more related to a mixed ion exchange/hydrophobic adsorption than to real covalent immobilization. Finally, we propose some possibilities that can permit not only the covalent immobilization of this enzyme, but also their stabilization via multipoint covalent attachment.

Papain is a cysteine protease from papaya, with many applications due to its broad specificity. This paper reviews for first time the immobilization of papain on different supports (organic, inorganic or hybrid supports) presenting some of the features of the utilized immobilization strategies (e.g., epoxide, glutaraldehyde, genipin, glyoxyl for covalent immobilization). Special focus is placed on the preparation of magnetic biocatalysts, which will permit the simple recovery of the biocatalyst even if the medium is a suspension. Problems specific to the immobilization of proteases (e.g., steric problems when hydrolyzing large proteins) are also defined. The benefits of a proper immobilization (enzyme stabilization, widening of the operation window) are discussed, together with some artifacts that may suggest an enzyme stabilization that may be unrelated to enzyme rigidification.

Papain is a cysteine endopeptidase of vegetal origin (papaya (Carica papaya L.) with diverse applications in food technology. In this review we have focused our attention on its application in the production of bio-peptides by hydrolysis of proteins from fish residues. This way, a residual material, that can become a contaminant if dumped without control, is converted into highly interesting products. The main bioactivity of the produced peptides is their antioxidant activity, followed by their nutritional and functional activities, but peptides with many other bioactivities have been produced. Thera are also examples of production of hydrolysates with several bioactivities. The enzyme may be used alone, or in combination with other enzymes to increase the degree of hydrolysis.

Papain is a cysteine protease from papaya, with many applications due to its broad specificity. This paper reviews for first time the immobilization of papain on different supports (organic, inorganic or hybrid supports) presenting some of the features of the utilized immobilization strategies (e.g., epoxide, glutaraldehyde, genipin, glyoxyl for covalent immobilization). Special focus is placed on the preparation of magnetic biocatalysts, which will permit the simple recovery of the biocatalyst even if the medium is a suspension. Problems specific to the immobilization of proteases (e.g., steric problems when hydrolyzing large proteins) are also defined. The benefits of a proper immobilization (enzyme stabilization, widening of the operation window) are discussed, together with some artifacts that may suggest an enzyme stabilization that may be unrelated to enzyme rigidification.

Papain is a cysteine endopeptidase of vegetal origin (papaya (Carica papaya L.) with diverse applications in food technology. In this review we have focused our attention on its application in the production of bio-peptides by hydrolysis of proteins from fish residues. This way, a residual material, that can become a contaminant if dumped without control, is converted into highly interesting products. The main bioactivity of the produced peptides is their antioxidant activity, followed by their nutritional and functional activities, but peptides with many other bioactivities have been produced. Thera are also examples of production of hydrolysates with several bioactivities. The enzyme may be used alone, or in combination with other enzymes to increase the degree of hydrolysis.

This review aims to cover the uses of the commercially available protease Alcalase in the production of biologically active peptides since 2010. Immobilization of Alcalase has also been reviewed, as immobilization of the enzyme may improve the final reaction design enabling the use of more drastic conditions and the reuse of the biocatalyst. That way, this review presents the production, via Alcalase hydrolysis of different proteins, of peptides with antioxidant, angiotensin I–converting enzyme inhibitory, metal binding, antidiabetic, anti-inflammatory and antimicrobial activities (among other bioactivities) and peptides that improve the functional, sensory and nutritional properties of foods. Alcalase has proved to be among the most efficient proteases for this goal, using different protein sources, being especially interesting the use of the protein residues from food industry as feedstock, as this also solves nature pollution problems. Very interestingly, the bioactivities of the protein hydrolysates further improved when Alcalase is used in a combined way with other proteases both in a sequential way or in a simultaneous hydrolysis (something that could be related to the concept of combi-enzymes), as the combination of proteases with different selectivities and specificities enable the production of a larger amount of peptides and of a smaller size.

Due to the problems raised by the use of animal or microbial recombinant proteases, the use of proteases from vegetable origin is becoming increasingly popular.. Among them, sulfidryl proteases have a special interest. Ficin is an outstanding example of this kind of proteases. This paper aims to be to make a comprehensive review of the recent uses of this enzyme, including for example protein hydrolysis, the production of bioactive peptides and antibodies fragments (researchers point that ficin results are more reproducible than using other proteases), meat tenderization, milk coagulations in cheese making or peptide synthesis. Efforts to get industrial immobilized biocatalysts of the enzyme will be also described. The review shows the huge potential and brilliant prospect that this enzyme can have in the near future.

This review aims to cover the uses of the commercially available protease Alcalase in the production of biologically active peptides since 2010. Immobilization of Alcalase has also been reviewed, as immobilization of the enzyme may improve the final reaction design enabling the use of more drastic conditions and the reuse of the biocatalyst. That way, this review presents the production, via Alcalase hydrolysis of different proteins, of peptides with antioxidant, angiotensin I–converting enzyme inhibitory, metal binding, antidiabetic, anti-inflammatory and antimicrobial activities (among other bioactivities) and peptides that improve the functional, sensory and nutritional properties of foods. Alcalase has proved to be among the most efficient proteases for this goal, using different protein sources, being especially interesting the use of the protein residues from food industry as feedstock, as this also solves nature pollution problems. Very interestingly, the bioactivities of the protein hydrolysates further improved when Alcalase is used in a combined way with other proteases both in a sequential way or in a simultaneous hydrolysis (something that could be related to the concept of combi-enzymes), as the combination of proteases with different selectivities and specificities enable the production of a larger amount of peptides and of a smaller size.

Due to the problems raised by the use of animal or microbial recombinant proteases, the use of proteases from vegetable origin is becoming increasingly popular.. Among them, sulfidryl proteases have a special interest. Ficin is an outstanding example of this kind of proteases. This paper aims to be to make a comprehensive review of the recent uses of this enzyme, including for example protein hydrolysis, the production of bioactive peptides and antibodies fragments (researchers point that ficin results are more reproducible than using other proteases), meat tenderization, milk coagulations in cheese making or peptide synthesis. Efforts to get industrial immobilized biocatalysts of the enzyme will be also described. The review shows the huge potential and brilliant prospect that this enzyme can have in the near future.

A simplified procedure for the preparation of immobilized beta-amylase using non-purified extract from fresh sweet potato tubers is established in this paper, using differently activated agarose supports. Beta-amylase glutaraldehyde derivative was the preparation with best features, presenting improved temperature and pH stability and activity. The possibility of reusing the amylase was also shown, when this immobilized enzyme was fully active for five cycles of use. However, immobilization decreased enzyme activity to around 15%. This seems to be mainly due to diffusion limitations of the starch inside the pores of the biocatalyst particles. A fifteen-fold increase in the Km was noticed, while the decrease of Vmax was only 30% (10.1 U mg−1 protein and 7.03 U mg−1 protein for free and immobilized preparations, respectively).