Stabilization of Immobilized Enzymes Against Water-Soluble Organic Cosolvents and Generation of Hyper-Hydrophilic Micro-Environments Surrounding Enzyme Molecules

Enzymes usually undergo rapid inactivation in the presence of organic media. In some cases, the mechanism is quite simple. For example, when an enzyme, fully dispersed and immobilized inside porous supports, is inactivated, at neutral p^H and moderate temperature, in the presence of medium-high concentrations of water-miscible organic cosolvents, the unique cause of inactivation is the interaction of the enzyme with cosolvent molecules and the only inactivating effect is the promotion of conformational changes on enzyme structure.

On this basis, two distinct strategies for stabilization of enzymes against organic solvents are proposed:

a. reduction of the causes of inactivation: generation of hyper-hydrophilic micro-environments having a very open structure and fully surrounding every enzyme molecule;

b. reduction of the effects of inactivation: “rigidification of enzymes” via multipoint covalent immobilization.

By using penicillin G acylase (PGA) as a model enzyme, both strategies have been evaluated and compared. Both stabilizing strategies had significant effects. In this case, hydrophilization of the enzyme nano-environment was found to be more effective than rigidification of the enzyme via multipoint covalent attachment. The combined effect of both stabilizing strategies was also tested: multipoint covalently immobilized enzyme molecules were completely surrounded by hyper-hydrophilic microenvironments. In this way, native PGA that was unstable against organic cosolvents (completely inactivated in less than 3 min in 95% dioxane) was transformed into a very stable immobilized derivative (preserving more than 80% of activity after 40 days under the same conditions).     

Stabilization of heterodimeric enzyme by multipoint covalent immobilization: Penicillin G acylase from Kluyvera citrophila

We have developed a strategy for immobilization-stabilization of penicillin G acylase (PGA) from Kluyvera citrophila by controlled multipoint covalent attachment to agarose-aldehyde gels. This enzyme is composed by two dissimilar subunits noncovalently bound. Thus, in this article we establish clear correlations between enzyme stabilization and the multipoint immobilization and/or between enzyme stabilization and the involvement of the two subunits in the attachment of them to the support. We have demonstrated that important thermal stabilizations of derivatives were only obtained through a very intense enzyme-support multipoint attachment involving the whole enzyme molecule. In this way, we have prepared derivatives preserving more than 90% of catalytic activity and being more than 1000-fold more stable than soluble and one-point attached enzyme. In addition, the involvement of the two subunits in the covalent attachment to the support has proved to be essential to develop interesting strategies for reactivation of inactivated enzyme molecules [e.g., by refolding of immobilized PGA after previous unfolding with urea and sodium dodecyl sulfate (SDS)]. (c) 1993 John Wiley & Sons, Inc.     

Stabilization of enzymes by multipoint immobilization of thiolated proteins on new epoxy-thiol supports

The controlled and partial modification of epoxy groups of Eupergit C and EP-Sepabeads with sodium sulfide has permitted the preparation of thiol-epoxy supports. Their use allowed not only the specific immobilization of enzymes through their thiol groups via thiol-disulfide interchange, but also enzyme stabilization via multipoint covalent attachment. Penicillin G acylase (PGA) from Escherichia coli and lipase from Rhizomucor miehei were used as model enzymes. Both enzymes lacked exposed cysteine residues, but were introduced via chemical modification under very mild conditions. In the first moments of the immobilization, a certain percentage of immobilized protein could be released from the support by incubation with DTT; this confirms that the first step was via a thiol-disulfide interchange. Moreover, the promotion of some further epoxy-enzyme bonds was confirmed because no enzyme release was detected after some immobilization time by incubation with DTT. In the case of the heterodimeric PGA, it was possible to demonstrate the formation of at least one epoxy bond per enzyme subunit by analyzing with SDS-PAGE the supernatants obtained after boiling the enzyme derivatives in the presence of mercaptoethanol and SDS. Thermal inactivation studies showed that these multipoint enzyme-support attachments promoted an increase in the stability of the immobilized enzymes. In both cases, the stabilization factor was around 12-15-fold comparing optimal derivatives with their just-thiol immobilized counterparts.     

Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint covalent attachment*

Epoxy supports (Eupergit C) may be very suitable to achieve the multipoint covalent attachment of proteins and enzymes, therefore, to stabilize their three-dimensional structure. To achieve a significant multipoint covalent attachment, the control of the experimental conditions was found to be critical. A three-step immobilization/stabilization procedure is here proposed: 1) the enzyme is firstly covalently immobilized under very mild experimental conditions (e.g. pH 7.0 and 20 degrees C); 2) the already immobilized enzyme is further incubated under more drastic conditions (higher pH values, longer incubation periods, etc.) to "facilitate" the formation of new covalent linkages between the immobilized enzyme molecule and the support; 3) the remaining groups of the support are blocked to stop any additional interaction between the enzyme and the support. Progressive establishment of new enzyme-support attachments was showed by the progressive irreversible covalent immobilization of several subunits of multi-subunits proteins (all non-covalent structures contained in crude extracts of different microorganism, penicillin G acylase and chymotrypsin). This multipoint covalent attachment enabled the significant thermostabilization of two relevant enzymes, (compared with the just immobilized derivatives): chymotrypsin (5-fold factor) and penicillin G acylase (18-fold factor). Bearing in mind that this stabilization was additive to that achieved by conventional immobilization, the final stabilization factor become 100-fold comparing soluble penicillin G acylase and optimal derivative. These stabilizations were observed also when the inactivations were promoted by the enzyme exposure to drastic pH values or the presence of cosolvents.     

Additional stabilization of penicillin G acylase-agarose derivatives by controlled chemical modification with formaldehyde.

We have tested the effect of chemical modifications with formaldehyde on the activity/stability of immobilized derivatives of the enzyme penicillin G acylase (PGA). These derivatives were previously stabilized through enzyme-support multipoint covalent attachment. We carried out very different chemical treatments of our derivatives by testing the effect of different variables which control the intensity and the nature of these amine-formaldehyde reactions. The variables tested were: formaldehyde concentration, pH, time, and temperature. We also developed a colorimetric titration of the free amine groups on immobilized PGA in order to evaluate the extension of the reaction between formaldehyde and the amine groups of the enzyme. As a consequence of these studies, we have been able to get additional stabilizations of our previously stabilized-immobilized derivatives: e.g. a factor of 24-fold was achieved in terms of stabilization against irreversible thermal inactivation. The integrated effect of additional chemical modification plus previous multipoint covalent attachment has allowed us to prepare PGA derivatives which are 50,000 more thermostable than native PGA as well as most of the commercial PGA derivatives.     

Immobilization of enzymes on heterofunctional epoxy supports

Immobilization of enzymes and proteins on activated supports permits the simplification of the reactor design and may be used to improve some enzyme properties. In this sense, supports containing epoxy groups seem to be useful to generate very intense multipoint covalent attachment with different nucleophiles placed on the surface of enzyme molecules (e.g., amino, thiol, hydroxyl groups). However, the intermolecular reaction between epoxy groups and soluble enzymes is extremely slow. To solve this problem, we have designed "tailor-made" heterofunctional epoxy supports. Using these, immobilization of enzymes is performed via a two-step process: (i) an initial physical or chemical intermolecular interaction of the enzyme surface with the new functional groups introduced on the support surface and (ii) a subsequent intense intramolecular multipoint covalent reaction between the nucleophiles of the already immobilized enzyme and the epoxy groups of the supports. The first immobilization may involve different enzyme regions, which will be further rigidified by multipoint covalent attachment. The design of some heterofunctional epoxy supports and the performance of the immobilization protocols are described here. The whole protocol to have an immobilized and stabilized enzyme could take from 3 days to 1 week.     

Epoxy sepabeads: a novel epoxy support for stabilization of industrial enzymes via very intense multipoint covalent attachment

Sepabeads-EP (a new epoxy support) has been utilized to immobilize-stabilize the enzyme penicillin G acylase (PGA) via multipoint covalent attachment. These supports are very robust and suitable for industrial purposes. Also, the internal geometry of the support is composed by cylindrical pores surrounded by the convex surfaces (this offers a good geometrical congruence for reaction with the enzyme), and it has a very high superficial density of epoxy groups (around 100 micromol/mL). These features should permit a very intense enzyme-support interaction. However, the final stability of the immobilized enzyme is strictly dependent on the immobilization protocol. By using conventional immobilization protocols (neutral pH values, nonblockage of the support) the stability of the immobilized enzyme was quite similar to that achieved using Eupergit C to immobilize the PGA. However, when using a more sophisticated three-step immobilization/stabilization/blockage procedure, the Sepabeads derivative was hundreds-fold more stable than Eupergit C derivatives. The protocol used was as follows: (i) the enzyme was first covalently immobilized under very mild experimental conditions (e.g., pH 7.0 and 20 degrees C); (ii) the already immobilized enzyme was further incubated under more drastic conditions (higher pH values, long incubation periods, etc.) in order to "facilitate" the formation of new covalent linkages between the immobilized enzyme molecule and the support; (iii) the remaining epoxy groups of the support were blocked with very hydrophilic compounds to stop any additional interaction between the enzyme and the support. This third point was found to be critical for obtaining very stable enzymes: derivatives blocked with mercaptoethanol were much less stable than derivatives blocked with glycine or other amino acids. This was attributed to the better masking of the hydrophobicity of the support by the amino acids (having two charges).     

Control of protein immobilization: coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance

Mutagenesis and immobilization are usually considered to be unrelated techniques with potential applications to improve protein properties. However, there are several reports showing that the use of site-directed mutagenesis to improve enzyme properties directly, but also how enzymes are immobilized on a support, can be a powerful tool to improve the properties of immobilized biomolecules for use as biosensors or biocatalysts. Standard immobilizations are not fully random processes, but the protein orientation may be difficult to alter. Initially, most efforts using this idea were addressed towards controlling the orientation of the enzyme on the immobilization support, in many cases to facilitate electron transfer from the support to the enzyme in redox biosensors. Usually, Cys residues are used to directly immobilize the protein on a support that contains disulfide groups or that is made from gold. There are also some examples using His in the target areas of the protein and using supports modified with immobilized metal chelates and other tags (e.g., using immobilized antibodies). Furthermore, site-directed mutagenesis to control immobilization is useful for improving the activity, the stability and even the selectivity of the immobilized protein, for example, via site-directed rigidification of selected areas of the protein. Initially, only Cys and disulfide supports were employed, but other supports with higher potential to give multipoint covalent attachment are being employed (e.g., glyoxyl or epoxy-disulfide supports). The advances in support design and the deeper knowledge of the mechanisms of enzyme-support interactions have permitted exploration of the possibilities of the coupled use of site-directed mutagenesis and immobilization in a new way. This paper intends to review some of the advances and possibilities that these coupled strategies permit.     

Promotion of multipoint covalent immobilization through different regions of genetically modified penicillin G acylase from E. coli

A novel approach is proposed to prepare a set of immobilized derivatives of a enzyme covalently rigidified through different regions of its surface. Six different variants of penicillin G acylase (PGA) from Escherichia coli (which lacks Cys) were prepared by introducing a unique Cys residue via site-directed mutagenesis in six different enzyme regions which were rich in Lys residues. All variants exhibited a similar activity and stability compared to those of the native enzyme. Each variant was immobilized on supports having a low concentration of reactive disulfide moieties and a high concentration of poorly reactive epoxy groups. After immobilization at pH 7.0 by site-directed thiol-disulfide intermolecular exchange, derivatives were further incubated at pH 10.0 for 48 h to promote an additional intramolecular reaction between Lys residues of enzyme and epoxy groups of the support. The establishment of at least three covalent attachments per PGA molecule was determined for all immobilized enzyme variants. The different derivatives exhibited diverse stability against several distorting agents and different selectivity in two interesting reactions. The derivative of the PGA variant obtained by replacement of GlnB380 by Cys was the most stable against heat and organic cosolvents: it preserved 90% of the initial activity and was 30-fold more stable than soluble PGA. This derivative also exhibited an improved enantioselectivity in the hydrolysis of chiral esters (E was improved from 8 to 16) and in kinetically controlled synthesis of amides (synthetic yields were increased from 31 to 49%).     

Site-directing an intense multipoint covalent attachment (MCA) of mutants of the Geobacillus thermocatenulatus lipase 2 (BTL2): Genetic and chemical amination plus immobilization on a tailor-made support

Immobilized enzymes are preferred over their soluble counterparts due to their robustness in harsh industrial processes; the most stable enzyme derivatives are often produced through multipoint covalent attachment (MCA). However, most enzymes are unable to establish optimal MCA to electrophile-type supports given the heterogeneous distribution and/or low content of primary amino groups on their surfaces; this restricts both the diversity of areas prone to react and the number of attachments to the support. To overcome this we propose combining site-directed immobilization and protein engineering to increase the number of bonds between a specific enzyme surface and a tailor-made support. We applied this novel strategy to engineered mutants of the lipase 2 from Geobacillus thermocatenulatus with one Cys exposed residue, that after genetic amination and/or chemical amination, were immobilized on glyoxyl-disulfide support using a site-directed MCA protocol. Two highly stabilized derivatives of chemically aminated lipase variants, in which site-directed MCA implied the surrounding surface of residues Cys344 or Cys40, were produced: the first one was 2.4-fold more productive than the reference derivative (648 g of hydrolyzed ester); the second derivative was 40% more selective (EPA/DHA molar ratio) and as active (1 μmol g catalyst⁻¹ min⁻¹) as the reference in the production of PUFAs.     

Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports

In this work, we have used supports activated with m-amino-phenylboronic groups to “reversibly” immobilize proteins under very mild conditions. Most of the proteins contained in a crude extract from E. coli could be immobilized on Eupergit C-250 L activated with phenylboronic and then fully desorbed from the support by using mannitol or SDS. This suggested that the immobilization of the proteins on these supports was not only via sugars interaction, but also by other interaction/s, quite unspecific, that might be playing a key role in the immobilization of the proteins. Penicillin acylase from E. coli (PGA) was also immobilized in Eupergit C activated with m-amino-phenylboronic groups. The enzyme could be fully desorbed with mannitol immediately after being immobilized on the support. However, longer incubation times of the immobilized preparation caused a reduction of protein elution from the boronate support in presence of mannitol. Moreover, these immobilized preparations showed a higher stability in the presence of organic solvents than the soluble enzyme; the stability also improved when the incubation time was increased (to a factor of 100). By desorbing the weakest bound enzyme molecules, it was possible to correlate adsorption strength with stabilization; therefore, it seems that this effect was due to the rigidification of the enzyme via multipoint attachment on the support.     

Heterofunctional supports for the one-step purification,immobilization and stabilization of large multimeric enzymes: Amino-glyoxyl versus amino-epoxy supports

For the immobilization-stabilization of multimeric enzymes, we propose a novel heterofunctional support containing a very low concentration of ionized amino groups and a very high concentration of very poorly reactive glyoxyl (aldehyde) groups. A large tetrameric enzyme, β-galactosidase from Thermus sp., was purified and dramatically stabilized with this novel support. The enzyme was first immobilized by physical adsorption via selective multipoint anionic exchange involving the largest region of the enzyme containing all enzyme subunits. Then, an additional long incubation of the immobilized derivative under alkaline conditions was performed in order to promote an intense intramolecular multipoint covalent attachment between amino groups of the adsorbed enzyme and the very stable glyoxyl groups on the support. This novel β-galactosidase derivative is the first one in which the four subunits of this enzyme become attached to a pre-existing support. Additionally, the novel amino-glyoxyl supports were much more suitable than amino-epoxy supports for intramolecular multipoint covalent immobilization of the adsorbed enzyme onto the support. In fact, at pH 7.0, the new supports covalently immobilize the physically adsorbed protein 24-fold more rapidly than epoxy supports. Furthermore, derivatives prepared on amino-glyoxyl supports preserved 85% of catalytic activity and were 5-fold more stable than derivatives prepared on amino-epoxy supports and more than 1000-fold more stable than soluble enzyme.     

Synthesis of sulfated bioactive peptides using immobilized arylsulfotransferase from Eubacterium sp.

Summary An arylsulfotransferase from Eubacterium sp. was immobilized on agarose gels by multipoint covalent attachment. The yield of immobilization was 80% with an activity of 11 UA/ml of derivative. After 19 days of incubation, the loss of activity of the derivative was 36%. The immobilized preparation was used to transfer selectively a sulfate group from p-nitrophenolsulfate to selected tyrosine containing biologically active peptides in 92–99% of yield.     

Multipoint covalent immobilization of microbial lipase on chitosan and agarose activated by different methods

In this work Candida antarctica lipase type B (CALB) was immobilized on agarose and chitosan. The influence of activation agents (glycidol, glutaraldehyde and epichlorohydrin) and immobilization time (5, 24 and 72 h) on hydrolytic activity, thermal and alkaline stabilities of the biocatalyst was evaluated. Protein concentration and enzymatic activity in the supernatant were determined during the immobilization process. More active derivatives were attained when the enzymatic extract was first purified through dialysis. The highest activities achieved were: for agarose-glyoxyl (with glycidol), 845 U/g of gel, after 72 h of immobilization; for chitosan-glutaraldehyde and agarose-glutaraldehyde, respectively, 1209 U/g of gel and 2716 U/g of gel, after 5 h of immobilization. Thermal stability was significantly increased, when compared to the soluble enzyme: 20-fold for agarose-glyoxyl (with glycidol)-CALB, 18-fold for chitosan-glutaraldehyde-CALB and 21-fold for agarose-glutaraldehyde. The best derivative, 58-fold more stable than the soluble enzyme, was obtained when CALB was immobilized on chitosan activated in two steps, using glycidol and glutaraldehyde, 72 h immobilization time. The stabilization degree of the derivative increased with the immobilization time, an indication that a multipoint covalent attachment between enzyme and the support had really occurred.     

Stabilization of a multimeric beta-galactosidase from Thermus sp. strain T2 by immobilization on novel heterofunctional epoxy supports plus aldehyde-dextran cross-linking

This work exemplifies the advantages of using a battery of new heterofunctional epoxy supports to immobilize enzymes. We have compared the performance of a standard Sepabeads-epoxy support with other Sepabeads-epoxy supports partially modified with boronate, iminodiacetic, metal chelates, and ethylenediamine in the immobilization of the thermostable beta-galactosidase from Thermus sp. strain T2 as a model system. Immobilization yields depended on the support, ranging from 95% using Sepabeads-epoxy-chelate, Sepabeads-epoxy-amino, or Sepabeads-epoxy-boronic to 5% using Sepabeads-epoxy-IDA. Moreover, immobilization rates were also very different when using different supports. Remarkably, the immobilized beta-galactosidase derivatives showed very improved but different stabilities after favoring multipoint covalent attachment by long-term alkaline incubation, the enzyme immobilized on Sepabeads-epoxy-boronic being the most stable. This derivative had some subunits of the enzyme not covalently attached to the support (detected by SDS-PAGE). This is a problem if the biocatalysts were to be used in food technology. The optimization of the cross-linking with aldehyde-dextran permitted the full stabilization of the quaternary structure of the enzyme. The optimal derivative was very active in lactose hydrolysis even at 70 degrees C (over 1000 IU/g), maintaining its activity after long incubation times under these conditions and with no risk of product contamination with enzyme subunits.     

Green coconut fiber: a novel carrier for the immobilization of commercial laccase by covalent attachment for textile dyes decolourization

Commercial laccase formulation was immobilized on modified green coconut fiber silanized with 3-glycidoxypropyltrimethoxysilane, aiming to achieve a cheap and effective biocatalyst. Two different strategies were followed: one point (pH 7.0) and multipoint (pH 10.0) covalent attachment. The influence of immobilization time on enzymatic activity and the final reduction with sodium borohydride were evaluated. The highest activities were achieved after 2?h of contact time in all situations. Commercial laccase immobilized at pH 7.0 was found to have higher activity and higher affinity to the substrate. However, the immobilization by multipoint covalent attachment improved the biocatalyst thermal stability at 50?°C, when compared to soluble enzyme and to the immobilized enzyme at pH 7.0. The Schiff's bases reduction by sodium borohydride, in spite of causing a decrease in enzyme activity, showed to contribute to the increase of operational stability through bonds stabilization. Finally, these immobilized enzymes showed high efficiency in the continuous decolourization of reactive textile dyes. In the first cycle, the decolourization is mainly due to dyes adsorption on the support. However, when working in successive cycles, the adsorption capacity of the support decreases (saturation) and the enzymatic action increases, indicating the applicability of this biocatalyst for textile wastewater treatment.     

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) as a potential industrial biocatalyst

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) [poly(GMA‐co‐EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low‐cost, easy‐to‐prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA‐co‐EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA‐co‐EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:43–53, 2016     

Immobilization-stabilization of alpha-chymotrypsin by covalent attachment to aldehyde-agarose gels

We have developed a strategy for immobilization-stabilization of alpha-chymotrypsin by multipoint covalent attachment of the enzyme, through its amino groups, to agarosealdehyde gels. We have studied the role of the main variables that control the intensity of these enzyme-support multi-interaction processes (surface density of aldehyde groups in the activated gel, contact time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives, etc.). In this way, we have prepared a number of very different chymotrypsinagarose derivatives. Our best derivatives, with the most intense multipoint attachment, were more stable than one-point attached derivatives and were more than 60,000-fold more stable than soluble enzyme in the absence of autolysis phenomena. In spite of the dramatic stabilization, the catalytic activity of these derivatives is little changed (they only lose 35% of intrinsic activity after this intense enzyme-support multi-interaction process). In addition, we have also demonstrated the very high capacity of 6% aldehyde-agarose gels to immobilize pure chymotrypsin (40 mg enzyme/mL catalyst). Furthermore, we have been able to establish a clear correlation between enzyme-support multipoint covalent attachment, stabilization against very different denaturing agents (heat, urea, organic cosolvents), and insensitivity of those immobilized chymotrypsin molecules to some activating agents.     

Immobilization of fuculose-1-phosphate aldolase from E. coli to glyoxal-agarose gels by multipoint covalent attachment

Recombinant fuculose 1-phosphate aldolase (FucA) from E. coli has been immobilized by multipoint covalent attachment to glyoxal-agarose gels. Experiments, varying the main parameters that control the immobilization process (surface density of aldehyde groups, temperature, pH), were carried out. An immobilization yield of 80-90% and FucA retained activity on immobilized derivative of 10-20% can be achieved when pH 10, 20°C and 200 µmoles cm^-3 of aldehyde groups was used. The observed activity loss in the immobilization process might be related to the fact that the complex quaternary structure of the enzyme could not be maintained. A short contact-time enzyme support is required to obtain high ratio of active to total immobilized enzyme.


A highly loaded derivative of immobilized FucA (65 AU cm^-3 of support) has been prepared to use in aldol condensation reactions. Reactions catalyzed by these aldolases involve the use of non-conventional media because of substrate solubility. For instance, the condensation of dihydroxyacetone phosphate (DHAP) and Z-amino-propanal, Z-(R)-alaninal and Z-(S)- alaninal in highly concentrated water-in-oil emulsions gave synthetic yields of 40, 25 and 29% respectively.