Structural differences of commercial and recombinant lipase B from Candida antarctica: An important implication on enzymes thermostability.

Lipase B from Candida antarctica (CalB) is the most widely used lipase, including in many industrial sectors, such as in biodiesel and pharmaceuticals production. CalB has been produced by heterologous expression using Pichia pastoris under PGK constitutive promoter (named LipB). Here, we have studied the structural features of commercial CalB and LipB enzymes using circular dichroism and fluorescence under different conditions. In the presence of denaturing agents CalB was more stable than LipB, in contrast, at increasing temperatures, LipB was more thermostable than CalB. Mass spectrometry data indicates that both enzymes have an insertion of amino acids related to α-factor yeast signal, however LipB enzyme showed the addition of nine residues at the N-terminal while CalB showed only four residues. Molecular modeling of LipB showed the formation of an amphipathic α-helix in N-terminal region that was not observed in CalB. This data suggests that this new α-helix possess could be involved in LipB thermostability. These results associated with new structural studies may provide information to the design of novel biocatalysts.

Immobilization of Lipases on Heterofunctional Octyl-Glyoxyl Agarose Supports: Improved Stability and Prevention of the Enzyme Desorption.

Lipases are among the most widely used enzymes in industry. Here, a novel method is described to rationally design the support matrix to retain the enzyme on the support matrix without leaching and also activate the enzyme for full activity retention. Lipases are interesting biocatalysts because they show the so-called interfacial activation, a mechanism of action that has been used to immobilize lipases on hydrophobic supports such as octyl-agarose. Thus, adsorption of lipases on hydrophobic surfaces is very useful for one step purification, immobilization, hyperactivation, and stabilization of most lipases. However, lipase molecules may be released from the support under certain conditions (high temperature, organic solvents), as there are no covalent links between the enzyme and the support matrix. A heterofunctional support has been proposed in this study to overcome this problem, such as the heterofunctional glyoxyl-octyl agarose beads. It couples the numerous advantages of the octyl-agarose support to covalent immobilization and creates the possibility of using the biocatalyst under any experimental conditions without risk of enzyme desorption and leaching. This modified support may be easily prepared from the commercially available octyl-agarose. Preparation of this useful support and enzyme immobilization on it via covalent linking is described here. The conditions are described to increase the possibility of achieving at least one covalent attachment between each enzyme molecule and the support matrix.

Antimicrobial peptides: promising compounds against pathogenic microorganisms.

In the last decades, the indiscriminate use of conventional antibiotics has generated high rates of microbial resistance. This situation has increased the need for obtaining new antimicrobial compounds against infectious diseases. Among these, antimicrobial peptides (AMPs) constitute a promising alternative as therapeutic agents against various pathogenic microbes. These therapeutic agents can be isolated from different organisms, being widespread in nature and synthesized by microorganisms, plants and animals (both invertebrates and vertebrates). Additionally, AMPs are usually produced by a non-specific innate immune response. These peptides are involved in the inhibition of cell growth and in the killing of several microorganisms, such as bacteria, fungi, enveloped viruses, protozoans and other parasites. They have many interesting properties as potential antibiotics, such as relatively small sizes (below 25-30 kDa), amphipathic structures, cationic nature, and offer low probability for the generation of microbial resistance. In recent years, many novel AMPs, with very promising therapeutic properties, have been discovered. These peptides have been the base for the production of chemical analogs, which have been designed, chemically synthesized and tested in vitro for their antimicrobial activity. This review is focused on antibacterial (against Gram (-) and Gram (+) bacteria) and antifungal peptides, discussing action mode of AMPs, and recent advances in the study of the molecular basis of their anti-microbial activity. Finally, we emphasize on their current pharmacological development, future directions and applications of AMPs as promising antibiotics of therapeutic use for microbial infections.

Use of enzymes in the production of semi-synthetic penicillins and cephalosporins: drawbacks and perspectives.

Semi-synthetic ß-lactamic antibiotics are the most used anti-bacteria agents, produced in hundreds tons/year scale. It may be assumed that this situation will even increase during the next years, with new ß-lactamic antibiotics under development. They are usually produced by the hydrolysis of natural antibiotics (penicillin G or cephalosporin C) and the further amidation of natural or modified antibiotic nuclei with different carboxylic acyl donor chains. Due to the contaminant reagents used in conventional chemical route, as well as the high energetic consumption, biocatalytic approaches have been studied for both steps in the production of these very interesting medicaments during the last decades. Recent successes in some of these methodologies may produce some significant advances in the antibiotics industry. In fact, the hydrolysis of penicillin G to produce 6-APA catalyzed by penicillin G acylase is one of the most successful historical examples of the enzymatic biocatalysis, and much effort has been devoted to find enzymatic routes to hydrolyze cephalosporin C. Initially this could be accomplished in a quite complex system, using a two enzyme system (D-amino acid oxidase plus glutaryl acylase), but very recently an efficient cephalosporin acylase has been designed by genetic tools. Other strategies, including metabolic engineering to produce other antibiotic nuclei, have been also reported. Regarding the amidation step, much effort has been devoted to the improvement of penicillin acylases for these reactions since 1960. New reaction strategies, continuous product extraction or new penicillin acylases with better properties have proven to be the key to have competitive biocatalytic processes. In this review, a critical discussion of these very interesting advances in the application of enzymes for the industrial synthesis of semi-synthetic antibiotics will be presented.

Influence of mass transfer limitations on the enzymatic synthesis of beta-lactam antibiotics catalyzed by penicillin G acylase immobilized on glioxil-agarose.

Mass transfer effects were investigated for the synthesis of ampicillin and amoxicillin, at pH 6.5 and 25 degrees C, catalyzed by penicillin G acylase immobilized on agarose. The influence of external mass transfer was analysed using different stirring rates, ranging form 200 to 800 rpm. Above 400 rpm, the film resistance may be neglected. Intra-particle diffusion limitation was investigated using biocatalysts prepared with different enzyme loads and agarose with different mean pore diameters. When agarose with 6, 8 and 10% of crosslinking were used, for the same enzyme load, substrates and products concentration profiles presented no expressive differences, suggesting pore diameter is not important parameter. An increase on enzyme load showed that when more than 90 IU of enzyme activity were used per mL of support, the system was influenced by intra-particle mass transfer. A reactive-diffusive model was used to estimate effective diffusivities of substrates and products.

Advances in the design of new epoxy supports for enzyme immobilization-stabilization.

Multipoint covalent immobilization of enzymes (through very short spacer arms) on support surfaces promotes a very interesting 'rigidification' of protein molecules. In this case, the relative positions of each residue of the enzyme involved in the immobilization process have to be preserved unchanged during any conformational change induced on the immobilized enzyme by any distorting agent (heat, organic solvents etc.). In this way, multipoint covalent immobilization should induce a very strong stabilization of immobilized enzymes. Epoxy-activated supports are able to chemically react with all nucleophile groups placed on the protein surface: lysine, histidine, cysteine, tyrosine etc. Besides, epoxy groups are very stable. This allows the performance of very long enzyme-support reactions, enabling us to get very intense multipoint covalent attachment. In this way, these epoxy supports seem to be very suitable to stabilize industrial enzymes by multipoint covalent attachment. However, epoxy groups exhibit a low intermolecular reactivity towards nucleophiles and hence the enzymes are not able to directly react with the epoxy supports. Thus a rapid physical adsorption of enzymes on the supports becomes a first step, followed by an additional rapid 'intramolecular' reaction between the already adsorbed enzyme and the activated support. In this situation, a suitable first orientation of the enzyme on the support (e.g. through regions that are very rich in nucleophiles) is obviously necessary to get a very intense additional multipoint covalent immobilization. The preparation of different 'generations' of epoxy supports and the design of different protocols to fully control the first interaction between enzymes and epoxy supports will be reviewed in this paper. Finally, the possibilities of a directed immobilization of mutated enzymes (change of an amino acid by cysteine on specific points of the protein surface) on tailor-made disulfide-epoxy supports will be discussed as an almost-ideal procedure to achieve very intense and very efficient rigidification of a desired region of industrial enzymes.

Purification, immobilization, and stabilization of a lipase from Bacillus thermocatenulatus by interfacial adsorption on hydrophobic supports.

A lipase from Bacillus thermocatenulatus (BTL2) cloned in E. coli has been purified using a very simple method: interfacial activation on a hydrophobic support followed by desorption with Triton. Only one band was detected by SDS-PAGE. The pure enzyme was immobilized using different methodologies. BTL2 adsorbed on a hydrophobic support (octadecyl-Sepabeads) exhibited a hyperactivation with respect to the soluble enzyme, whereas the other immobilized preparations suffered a slight decrease in the expressed activity. The soluble enzyme was very stable, but all immobilized preparations were much more stable than the soluble enzyme, the octadecyl-Sepabeads-BTL2 preparation being the most stable one in all conditions (high temperature or in the presence of organic cosolvents), maintaining 100% of the activity at 65 degrees C or 30% of dioxane and 45 degrees C after several days of incubation. The glyoxyl preparation, the second more stable, retained 80% of the initial activity after 2 days, respectively. The adsorption of this thermophilic lipase on octadecyl-Sepabeads permitted an increase in the optimal temperature of the enzyme of 10 degrees C.

Design of new immobilized-stabilized carboxypeptidase a derivative for production of aromatic free hydrolysates of proteins.

This paper presents stable carboxypeptidase A (CPA)-glyoxyl derivatives, to be used in the controlled hydrolysis of proteins. They were produced after immobilizing-stabilizing CPA on cross-linked 6% agarose beads, activated with low and high concentrations of aldehyde groups, and different immobilization times. The CPA-glyoxyl derivatives were compared to other agarose derivatives, prepared using glutaraldehyde as activation reactant. The most stabilized CPA-glyoxyl derivative was produced using 48 h of immobilization time and high activation grade of the support. This derivative was approximately 260-fold more stable than the soluble enzyme and presented approximately 42% of the activity of the soluble enzyme for the hydrolysis of long-chain peptides (e.g., cheese whey proteins previously hydrolyzed with immobilized trypsin and chymotrypsin) and of the small substrate N-benzoylglycyl-l-phenylalanine (hippuryl-l-Phe). These results were much better than those achieved using the conventional support, glutaraldehyde-agarose. Amino acid analysis of the products of the acid hydrolysis of CPA (both soluble and immobilized) showed that approximately four lysine residues were linked on the glyoxyl agarose beads, suggesting the existence of an intense multipoint covalent attachment between the enzyme and the support. The maximum temperature of hydrolysis was increased from 50 degrees C (soluble enzyme) to 70 degrees C (most stable CPA-glyoxyl derivative). The most stable CPA-glyoxyl derivative could be efficiently used in the hydrolysis of long-chain peptides at high temperature (e.g., 60 degrees C), being able to release 2-fold more aromatic amino acids (Tyr, Phe, and Trp) than the soluble enzyme, under the same operational conditions. This new CPA derivative greatly increased the feasibility of using this protease in the production of protein hydrolysates that must be free of aromatic amino acids.

Biotransformations catalyzed by multimeric enzymes: stabilization of tetrameric ampicillin acylase permits the optimization of ampicillin synthesis under dissociation conditions.

The importance of the stabilization of the quaternary structure of multimeric enzymes has been illustrated using a model reaction with great industrial relevance: the enzymatic synthesis of ampicillin from 6-amino penicillanic acid (6APA) and phenylglycine methyl ester (PGM) catalyzed by the tetrameric enzyme alpha-amino acid ester hydrolase from Acetobacter turbidans. The stabilization of the multimeric structure of the enzyme was achieved by multi-subunit immobilization of the enzyme followed by its further solid-phase chemical intersubunit cross-linking with polyfunctional macromolecules (dextran-aldehyde). This stabilized derivative has permitted the study of the reaction under conditions where nonstabilized enzyme molecules tended to dissociate (e.g., absence of phosphate ions). Synthetic yields improved from around 65%, under conditions where the nonstabilized derivative was stable, to around 85% in conditions where only the stabilized derivative could be utilized (40% methanol and absence of phosphate ions). When using high concentrations of PGM, a significant worsening of the reaction performance was detected with a significant decrease in the yields (below 55%, using 50 mM 6APA and PGM). This problem has been sorted out by using a fed-batch reaction system. By addition of PGM continuously to the reaction mixture (to maintain the concentration between 0.5 and 3 mM), 95% of 6-APA could be transformed to antibiotic (47.5 mM) by only using a 20% excess of acylating ester.

One-step purification, covalent immobilization, and additional stabilization of poly-His-tagged proteins using novel heterofunctional chelate-epoxy supports.

Epoxy supports covalently immobilize proteins following a two-step mechanism; that is, the protein is physically adsorbed and then the covalent reaction takes place. This mechanism has been exploited to combine the selectivity of metal chelate affinity chromatography with the covalent immobilization capacity of epoxy supports. In this way, it has been possible to accomplish, in a simple manner, the purification, immobilization, and stabilization of a poly-His-tagged protein. To fulfill this objective we developed a new kind of multifunctional epoxy support (chelate epoxy support [CES]), which was tested using a poly-His-tagged glutaryl acylase as a model protein (an alphabeta-heterodimeric enzyme of significant industrial interest). The selectivity of the immobilization in CES toward poly-His-tagged proteins was dependent to a large extent on the density and nature of the chelated metal. The highest selectivity was achieved by using low-density chelate groups (e.g., 5 micromol/g) and metals with a low affinity (e.g., Co). However, the rate of covalent immobilization of the protein by its reaction with the epoxy groups on the support significantly increased at alkaline pH values. The multipoint attachment to the CES also depended on the reaction time. The immobilization of both glutaryl acylase subunits was achieved by incubation of the enzyme derivative at pH 10 for 24 h, with the best enzyme derivative 100-fold more stable than the soluble enzyme. By taking advantage of the selectivity properties of the novel support, we were able to immobilize up to 30 mg of protein per gram of modified Eupergit 250 using either pure enzyme or a very crude enzyme extract.

Modulation of penicillin acylase properties via immobilization techniques: one-pot chemoenzymatic synthesis of Cephamandole from Cephalosporin C.

The modulation of penicillin G acylase (PGA) properties via immobilization techniques has been performed studying the acylation of 7-aminocephalosporanic acid with R-mandelic acid methyl ester. PGA from Escherichia coli, immobilized onto agarose activated with glycidol (glyoxyl-agarose), has been used for the design of a novel one-pot synthesis of Cephamandole in aqueous medium and without isolation of intermediates, through three consecutive biotransformations catalyzed by D-amino acid oxidase, glutaryl acylase and PGA.

Structural and functional stabilization of L-asparaginase via multisubunit immobilization onto highly activated supports.

A new protocol for the stabilization of the quaternary structure of multimeric enzymes has been attempted using as model enzyme (tetrameric) L-asparaginase from Escherichia coli. Such strategy is based upon multisubunit covalent immobilization of the enzyme onto activated supports (agarose-glutaraldehyde). Supports activated with different densities of reactive groups were used; the higher the density of groups, the higher the stabilization attained. However, because of the complexity of that enzyme, even the use of the highest densities of reactive groups was not enough to encompass all four subunits in the immobilization process. Therefore, a further chemical intersubunit cross-linking with aldehyde-dextran was pursued; these derivatives displayed a fully stabilized multimeric structure. In fact, boiling the modified enzyme derivative in the presence of sodium dodecyl sulfate and beta-mercaptoethanol did not lead to release of any enzyme subunit into the medium. Such a derivative, prepared under optimal conditions, retained ca. 40% of the intrinsic activity of the free enzyme and was also functionally stabilized, with thermostabilization enhancements of ca. 3 orders of magnitude when compared with its soluble counterpart. This type of derivative may be appropriate for extracorporeal devices in the clinical treatment of acute leukemia and might thus bring about inherent advantages in that all subunits are covalently bound to the support, with a longer half-life and a virtually nil risk of subunit release into the circulating blood stream.

Affinity chromatography of polyhistidine tagged enzymes. New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions.

New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal-chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant beta-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media. IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins: (a) more than 30% of the natural proteins contained in crude extracts from E. coli become adsorbed, in addition to our target recombinant protein, on these IMAC supports via multipoint weak adsorptions; (b) the multimeric poly-His tagged enzyme may become adsorbed via several poly-His tags belonging to different subunits. In this way, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole). The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions: (i) less than 2% of natural proteins contained in crude extracts are now adsorbed on these novel supports; and (ii) the target multimeric enzyme may be fully desorbed from the support using 60 mM imidazole. In spite of this dramatic reduction of multi-point interactions, this dextran coating hardly affects the rate of the one-point adsorption of poly-His tagged proteins (80% of the rate of adsorption compared to uncoated supports). Therefore, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface. However, the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface.

Coimmobilization of L-asparaginase and glutamate dehydrogenase onto highly activated supports.

In the present research work, production of coimmobilized derivatives of L-asparaginase and glutamate dehydrogenase was attempted. Comparison of immobilization of each enzyme independently with coimmobilization of the two enzymes unfolded important advantages of the latter, namely a decrease in the induction period (time before the maximum reaction rate is virtually achieved) and an increase in the maximum reaction rate. The effectiveness of the independent enzyme derivatives was low; however, it was enhanced by three-fold when the enzymes were coimmobilized onto the same agarose-glutaraldehyde support. Each supporting agarose bead may in fact be viewed as a nano-reactor with in situ reaction and separation (i.e. elimination of the ammonia formed), with the nanoenvironment surrounding each enzyme molecule being essentially devoid of steric hindrance.

Modulation of lipase properties in macro-aqueous systems by controlled enzyme immobilization: enantioselective hydrolysis of a chiral ester by immobilized Pseudomonas lipase.

Lipase from Pseudomonas fluorescens (PFL) has been immobilized by using different immobilization protocols. The catalytic behavior of the different PFL derivatives in the hydrolytic resolution of fully soluble (R,S) 2-hydroxy 4-phenyl butanoic acid ethyl ester (HPBE) in aqueous medium was analyzed. The soluble enzyme showed a significant but low enantioselectivity, hydrolyzing the S isomer more rapidly than the R-isomer (E = 7). The enzyme, immobilized via a limited attachment to a long and flexible spacer arm, showed almost identical activity and specificity to the soluble enzyme. However, other derivatives, e.g. PFL adsorbed on supports covered by hydrophobic moieties (octyl, decaoctyl), exhibited significant hyperactivation on immobilization (approximately 7-fold). Simultaneously, the enantioselectivity of the PFL-immobilized enzyme was significantly improved (from E = 7 to E = 80). By using such derivatives, almost pure R ester isomer (e.e. > 99%) has been obtained after 55% hydrolysis of the racemic mixture of a solution of 10% (w/v) (R,S) HPBE. The derivatives could be used for 10 cycles without any significant decrease in the activity of the biocatalyst.

A kinetic study of synthesis of amoxicillin using penicillin G acylase immobilized on agarose.

We present a kinetic model for the synthesis of amoxicillin from p-hydroxyphenylglycine methyl ester and 6-aminopenicillanic acid, catalyzed by penicillin G acylase immobilized on agarose, at 25 degrees C. Michaelis-Menten kinetic parameters (with and without inhibition) were obtained from initial velocity data (pH 7.5 and 6.5). Amoxicillin synthesis reactions were used to validate the kinetic model after checking mass transport effects. A reasonable representation of this system was achieved under some operational conditions, but the model failed under others. Nevertheless, it will be useful whenever a simplified model is required, e.g., in model-based control algorithms for the enzymatic reactor.

Essential role of the concentration of immobilized ligands in affinity chromatography: purification of guanidinobenzoatase on an ionized ligand.

Guanidinobenzoatase, a plasma protein with possible application as a 'tumor marker', has been fully purified by one-step affinity chromatography. The affinity matrix was prepared by 'controlled' immobilization of an enzyme inhibitor (agmatine) onto commercial agarose gels containing carboxyl moieties activated as N-hydroxysuccinimide esters. In this way, agmatine becomes immobilized through an amido bond and preserves an ionized guanidino moiety. Different matrices with different concentration of ligands were prepared in order to evaluate their properties as affinity supports. Interestingly, matrices with a very low concentration of immobilized ligands (2 microlmol/ml, corresponding to the modification of only 5% of active groups in the commercial resins) exhibited a low capacity for unspecific adsorption of proteins (as anion-exchange resins) and displayed also a high capacity for specific adsorption of our target protein. On the other hand, when affinity matrices possessed a moderate concentration of agmatine (10 micromol/ml of gel or higher), two undesirable phenomena were observed: (a) the matrix behaves as a very good anionic exchange support able to non-specifically adsorb most of plasma proteins and (b) the specific adsorption of our target protein becomes much lower. The latter phenomenon could be due to steric hindrances promoted by the interaction between each individual immobilized ligand and the corresponding binding pocket in the target protein. These hindrances could also be promoted by the presence of a fairly dense layer of immobilized ligands covering the support surface, thus preventing interactions between immobilized ligands and partially buried protein-binding pockets. In this way, a successful affinity purification (23.5% yield, x220 purification factor, a unique electrophoretic band) could be achieved by combination of three approaches: (i) the use of affinity matrices possessing a very low density of immobilized ligands, (ii) performing affinity adsorption at high ionic strength and (iii) performing specific desorption with substrates or substrate analogues.

Immobilization of functionally unstable catechol-2,3-dioxygenase greatly improves operational stability.

Thermophilic catechol 2,3-dioxygenase (EC 1.13.11.2) from Bacillus stearothermophilus has been immobilized on highly activated glyoxyl agarose beads. The enzyme could be fully immobilized at 4 degrees C and pH 10.05 with a high retention of activity (around 80%). Enzyme immobilized under these conditions showed little increase in thermostability compared with the soluble enzyme, but further incubation of immobilized enzyme at 25 degrees C and pH 10.05 for 3 h before borohydride reduction resulted in conjugates exhibiting a 100-fold increase in stability (c.f. the free enzyme). The stability of catechol 2,3-dioxygenase immobilized under these conditions was essentially independent of protein concentration whereas free enzyme was rapidly inactivated at low protein concentrations. An apparent stabilization factor of over 700-fold was recorded in the comparison of free and immobilized catechol 2,3-dioxygenases at protein concentrations of 10 µg/ml. Immobilization increased the 'optimum temperature' for activity by 20 degrees C, retained activity at substrate concentrations where the soluble enzyme was fully inactivated and enhanced the resistance to inactivation during catalysis. These results suggest that the immobilization of the enzyme under controlled conditions with the generation of multiple covalent links between the enzyme and matrix both stabilized the quaternary structure of the protein and increased the rigidity of the subunit structures.

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.

Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support-polyethylenimine composites.

New tailor-made anionic exchange resins have been prepared, based on films of large polyethylenimine polymers (e.g., MW 25,000) completely coating, via covalent immobilization, the surface of different porous supports (agarose, silica, polymeric resins). Most proteins contained in crude extracts from different sources have been very strongly adsorbed on them. Ionic exchange properties of such composites strongly depend on the size of polyethylenimine polymers as well as on the exact conditions of the covalent coating of the solids with the polymer. On the contrary, similar coating protocols yield similar matrices by using different porous supports as starting material. For example, 77% of all proteins contained in crude extracts from Escherichia coli were adsorbed, at low ionic strength, on the best matrices, and less than 15% of the adsorbed proteins were eluted from the support in the presence of 0.3 M NaCl. Under these conditions, 100% of the adsorbed proteins were eluted from conventional DEAE supports. Such polyethylenimine-support composites were also very suitable to perform very strong and nondistorting reversible immobilization of industrial enzymes. For example, lipase from Candida rugosa (CRL), beta-galactosidase from Aspergillus oryzae and D-amino acid oxidase (DAAO) from Rhodotorula gracilis, were adsorbed on such matrices in a few minutes at pH 7.0 and 4 degrees C. Immobilized enzymes preserved 100% of catalytic activity and remained fully immobilized in 0.2 M NaCl. In addition to that, CRL and DAAO were highly stabilized upon immobilization. Stabilization of DAAO, a dimeric enzyme, seems to be due to the involvement of both enzyme subunits in the ionic adsorption.