Switch off/switch on of a cysteinyl protease as a way to preserve the active catalytic group by modification with a reversible covalent thiol modifier: Immobilization of ficin on vinyl-sulfone activated supports.

The immobilization of ficin (a cysteinyl proteases) on vinyl sulfone agarose produced its almost full inactivation. It was observed that the incubation of the free and immobilized enzyme in ß-mercaptoethanol produced a 20 % of enzyme activity recovery, suggesting that the inactivation due to the immobilization could be a consequence of the modification of the catalytic Cys. To prevent the enzyme inactivation during the immobilization, switching off of ficin via Cys reaction with dipyridyl-disulfide was implemented, giving a reversible disulfide bond that produced a fully inactive enzyme. The switch on of ficin activity was implemented by incubation in 1 M ß-mercaptoethanol. Using this strategy to immobilize the enzyme on vinyl sulfone agarose beads, the expressed activity of the immobilized ficin could be boosted up to 80 %. The immobilized enzyme presented a thermal stabilization similar to that obtained using ficin-glyoxyl-agarose beads. This procedure may be extended to many enzymes containing critical Cys, to permit their immobilization or chemical modification.

Design of Artificial Enzymes Bearing Several Active Centers: New Trends, Opportunities and Problems.

Harnessing enzymes which possess several catalytic activities is a topic where intense research has been carried out, mainly coupled with the development of cascade reactions. This review tries to cover the different possibilities to reach this goal: enzymes with promiscuous activities, fusion enzymes, enzymes + metal catalysts (including metal nanoparticles or site-directed attached organometallic catalyst), enzymes bearing non-canonical amino acids + metal catalysts, design of enzymes bearing a second biological but artificial active center (plurizymes) by coupling enzyme modelling and directed mutagenesis and plurizymes that have been site directed modified in both or in just one active center with an irreversible inhibitor attached to an organometallic catalyst. Some examples of cascade reactions catalyzed by the enzymes bearing several catalytic activities are also described. Finally, some foreseen problems of the use of these multi-activity enzymes are described (mainly related to the balance of the catalytic activities, necessary in many instances, or the different operational stabilities of the different catalytic activities). The design of new multi-activity enzymes (e.g., plurizymes or modified plurizymes) seems to be a topic with unarguable interest, as this may link biological and non-biological activities to establish new combo-catalysis routes.

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.

The combination of covalent and ionic exchange immobilizations enables the coimmobilization on vinyl sulfone activated supports and the reuse of the most stable immobilized enzyme.

The coimmobilization of lipases from Rhizomucor miehei (RML) and Candida antarctica (CALB) has been intended using agarose beads activated with divinyl sulfone. CALB could be immobilized on this support, while RML was not. However, RML was ionically exchanged on this support blocked with ethylendiamine. Therefore, both enzymes could be coimmobilized on the same particle, CALB covalently using the vinyl sulfone groups, and RML via anionic exchange on the aminated blocked support. However, immobilized RML was far less stable than immobilized CALB. To avoid the discarding of CALB (that maintained 90% of the initial activity after RML inactivation), a strategy was developed. Inactivated RML was desorbed from the support using ammonium sulfate and 1% Triton X-100 at pH 7.0. That way, 5 cycles of RML thermal inactivation, discharge of the inactivated enzyme and re-immobilization of a fresh sample of RML could be performed. In the last cycle, immobilized CALB activity was still over 90% of the initial one. Thus, the strategy permits that enzymes can be coimmobilized on vinyl sulfone supports even if one of them cannot be immobilized on it, and also permits the reuse of the most stable enzyme (if it is irreversibly attached to the support).

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.

Stabilization of enzymes via immobilization: Multipoint covalent attachment and other stabilization strategies.

The use of enzymes in industrial processes requires the improvement of their features in many instances. Enzyme immobilization, a requirement to facilitate the recovery and reuse of these water-soluble catalysts, is one of the tools that researchers may utilize to improve many of their properties. This review is focused on how enzyme immobilization may improve enzyme stability. Starting from the stabilization effects that an enzyme may experience by the mere fact of being inside a solid particle, we detail other possibilities to stabilize enzymes: generation of favorable enzyme environments, prevention of enzyme subunit dissociation in multimeric enzymes, generation of more stable enzyme conformations, or enzyme rigidification via multipoint covalent attachment. In this last point, we will discuss the features of an "ideal" immobilization protocol to maximize the intensity of the enzyme-support interactions. The most interesting active groups in the support (glutaraldehyde, epoxide, glyoxyl and vinyl sulfone) will be also presented, discussing their main properties and uses. Some instances in which the number of enzyme-support bonds is not directly related to a higher stabilization will be also presented. Finally, the possibility of coupling site-directed mutagenesis or chemical modification to get a more intense multipoint covalent immobilization will be discussed.

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.

Effect of Tris Buffer in the Intensity of the Multipoint Covalent Immobilization of Enzymes in Glyoxyl-Agarose Beads.

Tris is an extensively used buffer that presents a primary amine group on its structure. In the present work trypsin, chymotrypsin and penicillin G acylase (PGA) were immobilized/stabilized on glyoxyl agarose in presence of different concentrations of Tris (from 0 to 20 mM). The effects of the presence of Tris during immobilization were studied analyzing the thermal stability of the obtained immobilized biocatalysts. The results indicate a reduction of the enzyme stability when immobilized in the presence of Tris. This effect can be observed in inactivations carried out at pH 5, 7, and 9 with all the enzymes assayed. The reduction of enzyme stability increased with the Tris concentration. Another interesting result is that the stability reduction was more noticeable for immobilized PGA than in the other immobilized enzymes, the biocatalysts prepared in presence of 20 mM Tris lost totally the activity at pH 7 just after 1 h of inactivation, while the reference at this time still kept around 61 % of the residual activity. These differences are most likely due to the homogeneous distribution of the Lys groups in PGA compared to trypsin and chymotrypsin (where almost 50% of Lys group are in a small percentage of the protein surface). The results suggest that Tris could be affecting the multipoint covalent immobilization in two different ways, on one hand, reducing the number of available glyoxyl groups of the support during immobilization, and on the other hand, generating some steric hindrances that difficult the formation of covalent bonds.

Effect of Concentrated Salts Solutions on the Stability of Immobilized Enzymes: Influence of Inactivation Conditions and Immobilization Protocol.

This paper aims to investigate the effects of some salts (NaCl, (NH4)2SO4 and Na2SO4) at pH 5.0, 7.0 and 9.0 on the stability of 13 different immobilized enzymes: five lipases, three proteases, two glycosidases, and one laccase, penicillin G acylase and catalase. The enzymes were immobilized to prevent their aggregation. Lipases were immobilized via interfacial activation on octyl agarose or on glutaraldehyde-amino agarose beads, proteases on glyoxyl agarose or glutaraldehyde-amino agarose beads. The use of high concentrations of salts usually has some effects on enzyme stability, but the intensity and nature of these effects depends on the inactivation pH, nature and concentration of the salt, enzyme and immobilization protocol. The same salt can be a stabilizing or a destabilizing agent for a specific enzyme depending on its concentration, inactivation pH and immobilization protocol. Using lipases, (NH4)2SO4 generally permits the highest stabilities (although this is not a universal rule), but using the other enzymes this salt is in many instances a destabilizing agent. At pH 9.0, it is more likely to find a salt destabilizing effect than at pH 7.0. Results confirm the difficulty of foreseeing the effect of high concentrations of salts in a specific immobilized enzyme.

Effect of amine length in the interference of the multipoint covalent immobilization of enzymes on glyoxyl agarose beads.

Trypsin, chymotrypsin, penicillin G acylase and ficin extract have been stabilized by immobilization on glyoxyl agarose, adding different aliphatic compounds bearing a primary amine group during the immobilization: ethyl amine, butyl amine, hexyl amine (at concentrations ranging from 0 to 20 mM) and octyl amine (from 0 to 10 mM) to analyze their effects on the immobilized enzyme stability. As expected, the presence of amines reduced the intensity of the enzyme-support multipoint covalent attachment, and therefore the enzyme stability. However, it is clear that this effect is higher using octyl amine for all enzymes (in some cases the enzyme immobilized in the presence of 10 mM octyl amine was almost inactivated while the reference kept over 50 % of the initial activity). This way, it seems that the most important effect of the presence of aminated compounds came from the generation of steric hindrances to the enzyme/support multi-reaction promoted by the ammines that are interacting with the aldehyde groups. In some instances, just 1 mM of aminated compounds is enough to greatly decrease enzyme stability. The results suggested that, if the composition of the enzyme extract is unknown, to eliminate small aminated compounds may be necessary to maximize the enzyme-support reaction.

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.

Ficin: A protease extract with relevance in biotechnology and biocatalysis.

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.

Use of glyoxyl-agarose immobilized ficin extract in milk coagulation: Unexpected importance of the ficin loading on the biocatalysts.

A protein extract obtained from fig tree (Ficus carica) latex containing ficin activity was immobilized on glyoxyl agarose. Different biocatalyst loadings were used (3, 10, 30 and 85 mg/g). When casein was used as substrate, the expressed activities were 60%, 58%, 41% and 14%, respectively, very likely due to casein diffusional limitations. As expected, an increase of the concentration of either free or immobilized ficin reduced the clotting time of casein solution and milk. However, maintaining the same amount of ficin, lowly loaded ficin biocatalysts were unable to produce the clotting neither of the casein solutions nor of the milk, while highly loaded catalysts produced a good aggregate. Performing the proteolytic milk treatment at 4 °C to prevent aggregation and them incubating the milk at 40 °C, the use of immobilized enzyme in milk clotting gave coagulum yields of 19%, 24% and 27% for the 10 mg/g, 30 mg/g and 85 mg/g immobilized ficin respectively, while free ficin gave a yield of around 20% under similar ficin concentrations.

Amination of ficin extract to improve its immobilization on glyoxyl-agarose: Improved stability and activity versus casein.

Ficin extract has been aminated using ethylenediamine and carbodiimide to transform all exposed carboxylic groups into amino groups, retaining around 80% of activity versus benzoyl-d,l-arginine p-nitroanilide hydrochloride (BANA) and 90% versus casein. This aminated enzyme was then immobilized on glyoxyl agarose beads. After optimization of the immobilization protocol (immobilization at pH 10 for just 1 h), the new biocatalyst was compared to that obtained using the non-aminated enzyme. Activity versus BANA was lower, but was higher versus casein. The new biocatalyst was more stable than the reference mainly at pH 7. The new biocatalyst permitted to have a more linear course and a higher hydrolysis yield of casein at 75 °C. Moreover, the activity of the new preparations was significantly higher than the reference or the free enzyme in 8 M urea, at pH 7 and 55 °C. The enzyme in an overloaded biocatalyst exhibited a much higher specific activity versus casein (75% of the low loaded biocatalysts) than the non-aminated enzyme (only 30%), suggesting a more appropriate enzyme orientation that decreased steric hindrances. Finally, the enzyme was reused for 5 cycles of casein hydrolysis at 40 °C and pH 7 without any decrease in enzyme activity.

Cooperativity of covalent attachment and ion exchange on alcalase immobilization using glutaraldehyde chemistry: Enzyme stabilization and improved proteolytic activity.

Alcalase was scarcely immobilized on monoaminoethyl-N-aminoethyl (MANAE)-agarose beads at different pH values (<20% at pH 7). The enzyme did not immobilize on MANAE-agarose activated with glutaraldehyde at high ionic strength, suggesting a low reactivity of the enzyme with the support functionalized in this manner. However, the immobilization is relatively rapid when using low ionic strength and glutaraldehyde activated support. Using these conditions, the enzyme was immobilized at pH 5, 7, and 9, and in all cases, the activity vs. Boc-Ala-ONp decreased to around 50%. However, the activity vs. casein greatly depends on the immobilization pH, while at pH 5 it is also 50%, at pH 7 it is around 200%, and at pH 9 it is around 140%. All immobilized enzymes were significantly stabilized compared to the free enzyme when inactivated at pH 5, 7, or 9. The highest stability was always observed when the enzyme was immobilized at pH 9, and the worst stability occurred when the enzyme was immobilized at pH 5, in agreement with the reactivity of the amino groups of the enzyme. Stabilization was lower for the three preparations when the inactivation was performed at pH 5. Thus, this is a practical example on how the cooperative effect of ion exchange and covalent immobilization may be used to immobilize an enzyme when only one independent cause of immobilization is unable to immobilize the enzyme, while adjusting the immobilization pH leads to very different properties of the final immobilized enzyme preparation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2768, 2019.

Further Stabilization of Alcalase Immobilized on Glyoxyl Supports: Amination Plus Modification with Glutaraldehyde.

Alcalase was immobilized on glyoxyl 4% CL agarose beads. This permitted to have Alcalase preparations with 50% activity retention versus Boc-l-alanine 4-nitrophenyl ester. However, the recovered activity versus casein was under 20% at 50 °C, as it may be expected from the most likely area of the protein involved in the immobilization. The situation was different at 60 °C, where the activities of immobilized and free enzyme became similar. The chemical amination of the immobilized enzyme or the treatment of the enzyme with glutaraldehyde did not produce any significant stabilization (a factor of 2) with high costs in terms of activity. However, the modification with glutaraldehyde of the previously aminated enzyme permitted to give a jump in Alcalase stability (e.g., with most than 80% of enzyme activity retention for the modified enzyme and less than 30% for the just immobilized enzyme in stress inactivation at pH 7 or 9). This preparation could be used in the hydrolysis of casein at pH 9 even at 67 °C, retaining around 50% of the activity after 5 hydrolytic cycles when the just immobilized preparation was almost inactive after 3 cycles. The modified enzyme can be reused in hydrolysis of casein at 45 °C and pH 9 for 6 cycles (6 h) without any decrease in enzyme activity.