JoséM. Guisán

Aldehyde-agarose gels as activated supports for immobilization-stabilization of enzymes

Abstract Aldehyde groups, moderately separated from support surfaces, are proposed as suitable active groups for developing strategies to insolubilize-stabilize enzymes by multipoint covalent attachment to activated preexistent supports. A method for preparation of glyoxyl-Sepharose CL gels, Ag-O-CH2-CHO, with very different surface densities of active groups, up to 17 aldehyde residues per 1000 A2 of gel surface, is presented. These activated gels are very stable, even in moderately alkaline media, e.g., half-life time of these aldehyde groups at pH 10, 25°C, is 12 days. Experiments of insolubilization of the enzyme Penicillin G acylase on these supports indicate that one point amine-aldehyde attachments are fast but quite reversible. However, the binding of the enzyme on the support can be dramatically stabilized by two-point enzyme-support attachment. These qualities, as well as the absence of steric hindrance for the amine-aldehyde chemical reaction, make this activation method very suitable for designing intense, but not distorting, enzyme-support multiinteraction processes.

Preparation of activated supports containing low pK amino groups. A new tool for protein immobilization via the carboxyl coupling method

A method for the preparation of new aminated agarose gels containing monoaminoethyl-N-aminoethyl structures, MANA-agarose gels, has been developed. These gels contain primary amino groups with a very low pK value (6.8). In addition to that, we have been able to prepare very highly activated gels (e.g., 10% agarose gels containing up to 200 mu Eq of primary amines per milliliter). These two properties make these activated supports suitable for performing novel and interesting methods for protein immobilizations via very mild carbodiimide activation of carboxy groups. For example, very effective coupling reactions can be performed at pH 5.0-6.0 in the presence of low concentrations of activating agent, e.g., 1 mM. By using a model industrial enzyme, beta-galactosidase from Aspergillus oryzae, we have been able to demonstrate the excellent prospects of these novel activated supports.

Immobilization-stabilization of enzymes; variables that control the intensity of the trypsin (amine)-agarose (aldehyde) multipoint attachment

Abstract We have developed a strategy for immobilization-stabilization of trypsin by multipoint covalent attachment to agarose (aldehyde) gels. We have studied the role of four main variables that control the intensity of the trypsin (amine)-agarose (aldehyde) multiinteraction processes: (a) surface density of aldehyde groups in the activated gels, (b) pH of the multiinteraction medium, (c) contact time between insolubilized enzyme and activated support prior to borohydride reduction of the derivatives, and (d) temperature. Different combinations of these four variables have been tested to prepare a number of trypsin-agarose derivatives. All these derivatives preserved 100% of catalytic activity but showed very different stability values. The less stable derivative had exactly the same stability of soluble trypsin in the absence of autolysis phenomena. On the other hand, the three-dimensional structure of the most stable derivative was 5000-fold more stable than the one corresponding to unmodified trypsin. Amino acid analysis of hydrolysates of this very stable derivative reveals that seven lysine residues per trypsin molecule have reacted with the activated support during the process of preparation of the derivative.

Stabilization of enzymes by multipoint covalent attachment to agarose-aldehyde gels. Borohydride reduction of trypsin-agarose derivatives

Abstract The process of borohydride reduction of one-point-attached and multipoint-attached trypsin (amine)-agarose (aldehyde) derivatives has been studied. We have tested the effect of different variables that control the intensity of this reduction process on the activity and stability of the resulting derivatives. In this way, we have been able to establish the optimal conditions for this process: 1 mg ml −1 borohydride, pH 10.0, 25°C, 30 min of reaction time, and presence of benzamidine (competitive inhibitor of trypsin). In these conditions, the reduction of Schiffs bases formed between the enzyme and the support is very intense, the remaining aldehyde groups on the support are reduced completely, and the deleterious effects of borohydride on trypsin structure are negligible. We have also studied these deleterious effects of borohydride when reductions were performed in more drastic experimental conditions. A broad range of experimental conditions seems to be useful to test the reduction of a broad spectrum of multipoint-attached enzyme (amine)-support (aldehyde) derivatives.

Strategies for enzyme stabilization by intramolecular crosslinking with bifunctional reagents

Abstract A original strategy to obtain intramolecular crosslinking is discussed. This strategy consisted of three consecutive steps to direct the reaction to the formation of intramolecular crosslinks: (a) enzyme are partially modified with the bifunctional reagent in a very controlled fashion; (b) the excess of reagent is removed; and (c) the modified enzyme is incubated long-term to allow a crosslinking reaction without the competition of additional single-point modifications. In this way, we were able to obtain interesting stabilizations of immobilized derivatives of penicillin G acylase using glutaraldehyde as a crosslinking reagent under very controlled conditions.

Facile synthesis of artificial enzyme nano-environments via solid-phase chemistry of immobilized derivatives: Dramatic stabilization of penicillin acylase versus organic solvents

Abstract A method to generate a highly hydrophilic nano-environment surrounding immobilized enzymes [penicillin acylase (PGA)] has been developed. The enzyme was firstly multipoint immobilized on a highly activated support having an internal morphology composed by large hydrophilic surfaces (a). After irreversible enzyme immobilization, a high molecular weight polyamine molecule was further immobilized on the same support surface. In this way, all areas of the enzyme next to the support surface may become embedded in a hydrophilic environment (b); or the immobilized enzyme was modified with a high molecular weight poly-aldehydic polymer which also becomes a hydrophilic poly-hydroxyl macromolecule after borohydride reduction (c). The single application of each distinct (b or c) modifications did not promote stabilizing effects on immobilized PGA; however, the combined effect of both consecutive modifications (the chemical modification of the coimmobilized enzyme-polyamine derivative) promoted a dramatic stabilization of PGA versus organic solvents associated with a minimal loss of catalytic activity. The stabilizing effect was higher when the enzyme was incubated with large and hydrophobic organic solvents; thus, the modified derivative retained 80% of activity relative to soluble enzyme, but it was 1,000-fold more stable than the unmodified one in the presence of 90% tetraglyme. A fairly complete embedding of the whole enzyme surface in a highly hydrophilic nano-environment seems to be achieved.

Enzyme reaction engineering: synthesis of antibiotics catalysed by stabilized penicillin G acylase in the presence of organic cosolvents.

By using very active and very stable penicillin G acylase (PGA)--agarose derivatives we have studied the industrial design of equilibrium-controlled synthesis of lactamic antibiotics. In the presence of high concentrations of organic cosolvents we have carried out the direct enzymatic condensation of phenylacetic acid and 6-aminopenicillanic acid to yield the model antibiotic penicillin G. We have mainly studied the integrated effect of different variables that define the reaction medium on a number of parameters of industrial interest:time course of antibiotic synthesis, highest synthetic yields, stability of the catalyst, and solubility and stability of substrates and products. The main variables tested were the nature and concentration of the organic cosolvent, pH, and temperature. The effects of the variables tested on different parameters were quite different and sometimes opposite. Hence, the optimal experimental conditions for antibiotic synthesis catalysed by PGA were established, as a compromise solution, in order to obtain good values for every parameter of industrial interest. These conditions seem to be important parameters for scale-up (e.g. we have been able to reach more than 95% of synthetic yields with productivities around 0.5 tons of model antibiotic per year per liter of catalyst).

The presence of methanol exerts a strong and complex modulation of the synthesis of different antibiotics by immobilized penicillin G acylase

Abstract The yields in the synthesis of antibiotics catalyzed by Penicillin G acylase under very mild experimental conditions by using esters of side chains as acylating agents depend on the ratio between synthetase and hydrolase activities of penicillin G acylase. The presence of low concentrations of methanol in the reaction medium exerts complex and interesting changes in the catalytic properties of immobilized penicillin G acylase. When phenylglycine methyl ester is used as acylating agent, the presence of methanol has a double effect. It increased the ratio synthesis/hydrolysis of antibiotic, but decreased the ratio between synthesis of antibiotic and direct hydrolysis of the acyl donor ester. In this way, synthetic yields are increased when using an excess of acylating substrate (e.g., from 63% to 73%) because the key point in this reaction is to decrease the hydrolysis of the already synthesized antibiotic. When using an excess of antibiotic nucleus, the hydrolysis of antibiotic is strongly inhibited and then the ratio synthesis of antibiotic/hydrolysis of the acyl donor is the key point and the yields suffered a decrease (e.g., from 90 to 60%). On the contrary, when using mandelic acid methyl ester as acylating agent, methanol increases both ratios (hydrolysis/synthesis of the antibiotic and synthesis of antibiotic/hydrolysis of the acyl donor); thus, the yields increased by almost a twofold factor (e.g., from 38 to 70%). Finally, the contradictory effects observed in the presence of methanol can be modulated by using different enzyme derivatives. In fact, the effect of methanol on the Penicillin G acylase structure could be cleared out by increasing the stability of the enzyme derivative or by “fixing” the enzyme conformation in the presence of the organic solvent during the immobilization process.

Reversible and Strong Immobilization of Proteins by Ionic Exchange on Supports Coated with Sulfate-Dextran

New and strong ionic exchange resins have been prepared by the simple and rapid ionic adsorption of anionic polymers (sulfate‐dextran) on porous supports activated with the opposite ionic group (DEAE/MANAE). Ionic exchange properties of such composites were strongly dependent on the size of the ionic polymers as well as on the conditions of the ionic coating of the solids with the ionic polymers (optimal conditions were 400 mg of sulfate‐dextran 5000 kDa per gram of support). Around 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans could be adsorbed on these porous composites even at pH 7. This interaction was stronger than that using conventional carboxymethyl cellulose (CMC) and even others such as supports coated with aspartic‐dextran polymer. By means of the sequential use of the new supports and supports coated with polyethyleneimine (PEI), all proteins from crude extracts could be immobilized. In fact, a large percentage (over 50%) could be immobilized on both supports. Finally, some industrially relevant enzymes (β‐galactosidases from Aspergillus oryzae, Kluyveromyces lactis, and Thermus sp. strain T2, lipases from Candida antarctica A and B, Candida rugosa, Rhizomucor miehei, and Rhyzopus oryzae and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recoveries and immobilization rates. After enzyme inactivation, the protein could be fully desorbed from the support, and then the support could be reused for several cycles. Moreover, in some instances the enzyme stability was significantly improved, mainly in the presence of organic solvents, perhaps as a consequence of the highly hydrophilic microenvironment of the support.

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.