A review on the immobilization of bromelain.

This review shows the endeavors performed to prepare immobilized formulations of bromelain extract, usually from pineapple, and their use in diverse applications. This extract has a potent proteolytic component that is based on thiol proteases, which differ depending on the location on the fruit. Stem and fruit are the areas where higher activity is found. The edible origin of this enzyme is one of the features that determines the applications of the immobilized bromelain to a more significant degree. The enzyme has been immobilized on a wide diversity of supports via different strategies (covalent bonds, ion exchange), and also forming ex novo solids (nanoflowers, CLEAs, trapping in alginate beads, etc.). The use of preexisting nanoparticles as immobilization supports is relevant, as this facilitates one of the main applications of the immobilized enzyme, in therapeutic applications (as wound dressing and healing components, antibacterial or anticancer, mucus mobility control, etc.). A curiosity is the immobilization of this enzyme on spores of probiotic microorganisms via adsorption, in order to have a perfect in vivo compatibility. Other outstanding applications of the immobilized enzyme are in the stabilization of wine versus haze during storage, mainly when immobilized on chitosan. Curiously, the immobilized bromelain has been scarcely applied in the production of bioactive peptides.

Peptides with biological and technofunctional properties produced by bromelain hydrolysis of proteins from different sources: A review.

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.

A review on the immobilization of pepsin: A Lys-poor enzyme that is unstable at alkaline pH values.

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.

Effect of heat treatment on oxalate and hydrocyanic acid levels of malanga corms of two cultivars (Xanthosoma sagittifolium and Colocasia esculenta) in a murine model.

Xanthosoma sagittifolium and Colocasia esculenta contain high levels of nutrients; but have naturally toxic compounds, oxalates and hydrocyanic acid (HCN). The objective of this work was to evaluate the effect of heat treatment on the concentration of antinutrients in malanga corms and its effect on mice. Malanga samples were heated to a boil for 0 to 120 min; oxalates and HCN were determined by spectrophotometry, at 710 and 510 nm, respectively. Pellets were prepared from raw malanga flour (15 and 50%), cooked malanga (15 and 50%) and wheat flour (control) and fed for nine weeks to five groups of six mice each. Cooking of X. sagittifolium corms for 80 min reduced oxalates present by 75% (143 to 35.6 mg/100 g sample), while oxalates in C. esculenta were reduced by 83% (345 to 57.8 mg/100 g sample). HCN levels became negligible after 20 min of cooking. During the nine weeks of feeding the different mice groups showed no significant difference (p > 0.05) between initial and final weight, with respect of the control; mice did not lose their appetite. The results indicate that the consumption of cooked malanga does not pose an evident risk to health, assessed by the reduced level of antinutrients, being an excellent alternative for feeding people in communities with prevalence of food insecurity.

Immobilization of papain: A review.

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.

Bioactive peptides from fisheries residues: A review of use of papain in proteolysis reactions.

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.

Use of Alcalase in the production of bioactive peptides: A review.

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.

Relevance of substrates and products on the desorption of lipases physically adsorbed on hydrophobic supports.

Lipase B from Candida antarctica (CALB) has been physically immobilized on octyl-agarose via interfacial activation. The incubation of the enzyme in 80% ethanol at pH 5 and 25°C has not significant effect on enzyme activity. Moreover, the hydrolysis of 100mM tributyrin catalyzed by this biocatalyst exhibited a quite linear reaction course. However, a new cycle of tributyrin hydrolysis showed a drastic drop in the activity. SDS-PAGE gels of the supernatant and the biocatalyst showed a significant enzyme desorption after the reaction. Similar results could be appreciated using triacetin or sunflower oil, while using 300mM methyl phenyl acetate, butyl butyrate or ethyl butyrate most enzyme molecules remained immobilized. The results show that the detergent properties of some reaction products increase the enzyme release from the hydrophobic support, and this problem increased if the concentration of the reactants increased. Using 500mM tributyrin, even in fully aqueous medium, some enzyme desorption from the support may be observed. Thus, the results show a limitation of this kind of biocatalysts that should be considered in the selection of an industrial lipase biocatalyst.

Stabilization of Candida antarctica Lipase B (CALB) Immobilized on Octyl Agarose by Treatment with Polyethyleneimine (PEI).

Lipase B from Candida antarctica (CALB) was immobilized on octyl agarose (OC) and physically modified with polyethyleneimine (PEI) in order to confer a strong ion exchange character to the enzyme and thus enable the immobilization of other enzymes on its surface. The enzyme activity was fully maintained during the coating and the thermal stability was marginally improved. The enzyme release from the support by incubation in the non-ionic detergent Triton X-100 was more difficult after the PEI-coating, suggesting that some intermolecular physical crosslinking had occurred, making this desorption more difficult. Thermal stability was marginally improved, but the stability of the OCCALB-PEI was significantly better than that of OCCALB during inactivation in mixtures of aqueous buffer and organic cosolvents. SDS-PAGE analysis of the inactivated biocatalyst showed the OCCALB released some enzyme to the medium during inactivation, and this was partially prevented by coating with PEI. This effect was obtained without preventing the possibility of reuse of the support by incubation in 2% ionic detergents. That way, this modified CALB not only has a strong anion exchange nature, while maintaining the activity, but it also shows improved stability under diverse reaction conditions without affecting the reversibility of the immobilization.