Medicine

The modulation, via immobilization and engineering the reaction medium, of the enantioselectivity exhibited by the lipase from Candida antarctica B (CAL-B) in the hydrolysis of a-hydroxy-phenylacetic acid derivatives is shown. The enzyme was purified and immobilized using different protocols to obtain immobilized enzyme preparations with different orientations and micro-environments. The catalytic properties (activity, specificity, enantioselectivity) of the resulting derivatives were found to be quite different from each other. The enantioselectivity (E value) strongly depends on the type of derivative and the conditions employed. Thus, the enzyme immobilized on cyanogen bromide (CNBr) presented E=7.4, while the PEI derivative yielded E=67 in the hydrolysis of a-hydroxy-phenylacetic acid methyl ester under similar conditions. Moreover, the enantioselectivity of the PEI derivative decreased from 67 to 14 on lowering the reaction temperature from 25 to 4°C at pH 5, while the E of some other derivatives improved significantly under similar experimental changes. Similar changes in the E values were observed in the hydrolysis of (RS)-2-butyroyl-2-phenylacetic acid. Using this substrate, the interfacially adsorbed enzyme (octadecyl) afforded an E value of only 2 at pH 5, while the glutaraldehyde derivative presented a high enantioselectivity (E >400) under all conditions studied. The corresponding (S)-ester and (R)-acid were obtained with excellent enantiomeric excess using the glutaraldehyde derivative, while using the interfacially immobilized one there was no appreciable enantioselectivity. Thus, using differently immobilized derivatives and different experimental conditions, lipase enantioselectivity could vary from negligible to up to 400. The experimental conditions were also found to have varying effects on the different lipase derivatives.

A successful strategy for the immobilization of rennet from Mucor miehei has been developed. The strategy is based on the immobilization of the enzyme, via their sugar chains at high ionic strength on aminated supports having primary amino groups with a very low pK value. The rennet was covalently immobilized via sugar chains (previously oxidized with periodate), which act as natural spacer arms and allow a very high percentage of rennet activity to be kept against small (H-Leu-Ser-p-nitro-Phe-Nle-Ala-Leu-OMe.TFA (98%)) and macromolecular substrates (k-casein) (78%). The use of tailor-made aminated support was critical to obtain good stability values, because using fully aminated supports achieved much lower thermostability values than using 50% aminated supports. The optimized derivative was utilized to hydrolyze casein in milk. To prevent the coagulation of the milk in the presence of the derivative, the reaction was performed at 4°C (where hydrolyzed casein did not precipitate). Then the hydrolyzed milk was filtered and latter on heated to 30°C, achieving a similar aggregate to the one achieved with soluble rennet.

A single bond covalent immobilization of aminated DNA probes on magnetic particles suitable for selective molecular hybridization of traces of DNA samples has been developed. Commercial superparamagnetic nanoparticles containing amino groups were activated by coating with a hetero-functional polymer (aldehyde-aspartic-dextran). This new immobilization procedure provides many practical advantages: (a) DNA probes are immobilized far from the support surface preventing steric hindrances; (b) the surface of the nanoparticles cannot adsorb DNA ionically; (c) DNA probes are bound via a very strong covalent bond (a secondary amine) providing very stable immobilized probes (at 100 • C, or in 70% formamide, or 0.1N NaOH). Due to the extreme sensitivity of this purification procedure based on DNA hybridization, the detection of hybridized products could be coupled to a PCR-ELISA direct amplification of the DNA bond to the magnetic nanoparticles. As a model system, an aminated DNA probe specific for detecting Hepatitis C Virus cDNA was immobilized according to the optimised procedure described herein. Superparamagnetic nanoparticles containing the immobilized HCV probe were able to give a positive result after PCR-ELISA detection when hybridized with 1 mL of solution containing 10 −18 g/mL of HCV cDNA (two molecules of HCV cDNA). In addition, the detection of HCV cDNA was not impaired by the addition to the sample solution of 2.5 million-fold excess of non-complementary DNA. The experimental data supports the use of magnetic nanoparticles containing DNA probes immobilized by the procedure here described as a convenient and extremely sensitive procedure for purification/detection DNA/RNA from biological samples. The concentration/purification potential of the magnetic nanoparticles, its stability under a wide range of conditions, coupled to the possibility of using the particles directly in amplification by PCR greatly reinforces this methodology as a molecular diagnostic tool.

It has been found that lipase from Pseudomonas fluorescens (PFL) is able to aggregate into bimolecular structures (MW around 66 kD) even at moderate enzyme concentrations. At very low enzyme concentrations and in the presence of detergents, the same enzyme displayed a unimolecular structure with a molecular weight of 33 kD. Both enzyme structures displayed different functional properties. First, the bimolecular structure was much more stable than the unimolecular species (the bimolecular structure maintained over 80% of initial activity after 72 hours at 45°C, while the unimolecular structure retained only around 30% of initial activity after 4 hours of incubation under the same experimental conditions); and the bimolecular form presented a higher optimal T. Second, the unimolecular form showed a much lower K M for ethyl butyrate than the bimolecular form. Third, the interfacial activation in biphasic substrate-aqueous milieu was higher for the bimolecular form. Fourth, the unimolecular structure was less active but much more enantioselective than the unimolecular species in the model reaction used. It is proposed that the bimolecular aggregates of PFL might be formed by two open lipase molecules (mutual interfacial activation), in intimate contact, and that the bimolecular form represents an example of "pseudo-quaternary" structure.

The properties of a new and commercially available amino-epoxy support (amino-epoxy-Sepabeads) have been compared to conventional epoxy supports to immobilize enzymes, using the -galactosidase from Aspergillus oryzae as a model enzyme. The new support has a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. This support has both a great anionic exchanger strength and a high density of epoxy groups. Epoxy supports require the physical adsorption of the proteins onto the support before the covalent binding of the enzyme to the epoxy groups. Using conventional supports the immobilization rate is slow, because the adsorption is of hydrophobic nature, and immobilization must be performed using high ionic strength (over 0.5 M sodium phosphate) and a support with a fairly hydrophobic nature. Using the new support, immobilization may be performed at moderately low ionic strength, it occurs very rapidly, and it is not necessary to use a hydrophobic support. Therefore, this support should be specially recommended for immobilization of enzymes that cannot be submitted to high ionic strength. Also, both supports may be expected to yield different orientations of the proteins on the support, and that may result in some advantages in specific cases. For example, the model enzyme became almost fully inactivated when using the conventional support, while it exhibited an almost intact activity after immobilization on the new support. Furthermore, enzyme stability was significantly improved by the immobilization on this support (by more than a 12-fold factor), suggesting the promotion of some multipoint covalent attachment between the enzyme and the support (in fact the enzyme adsorbed on an equivalent cationic support without epoxy groups was even slightly less stable than the soluble enzyme).

Invertase from S. cerevisiae has been immobilized by ionic adsorption on Sepabeads fully coated with PEI. The enzyme was strongly adsorbed on the support (no desorption of the invertase was found under conditions in which all of the enzyme was released from conventional anionic exchanger supports (e.g., DEAE-agarose)). Nevertheless, the enzyme could still be desorbed after its inactivation, and new fresh enzyme could be adsorbed on the supports without detrimental effects on enzyme loading. This is a multimeric enzyme, its minimal oligomerization active state being the dimer, but under certain conditions of pH and concentration it may give larger multimers. Very interestingly, results suggested that the adsorption of the enzyme on this large and flexible polymeric bed was able to freeze some of the different oligomeric structures of the enzyme. Thus, we have found that the enzyme immobilized at certain pH values (pH 8.5) and high enzyme concentration, in which the main enzyme structure is the tetramer, was more stable than immobilized preparations produced in conditions under which oligomerization was not favorable (dimers at low enzyme concentration) or it was too high (e.g., hexamers-octamers at low pH value). The optimal enzyme preparation remained fully active after a 15-day incubation at 50°C and pH 4.5 (conditions of standard industrial use) and presented an optimal temperature approximately 5°C higher than that of soluble enzyme.

New tailor-made cationic exchange resins have been prepared by covalently binding aspartic-dextran polymers (e.g. MW 15 000-20 000) to porous supports (aminated agarose and Sepabeads). More than 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans have been strongly adsorbed on these porous materials at pH 5. This interaction was stronger than in conventional carboxymethyl cellulose (e.g., at pH 7 and 25°C, all proteins previously adsorbed at pH 5 were released from carboxymethyl cellulose, whereas no protein was released from the new supports under similar conditions). Ionic exchange properties of such composites were strongly dependent on the size of the aspartic-dextran polymers as well as on the exact conditions of the covalent coating of the solids with the polymer (optimal conditions: 100 mg aspartic-dextran 20 000/(mL of support); room temperature). Finally, some industrially relevant enzymes (Kluyveromices lactis, Aspergillus oryzae, and Thermus sp. -galactosidases, Candida antarctica B lipase, and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recovery and immobilization rates. After enzyme inactivation, the enzyme can be fully desorbed from the support and the support could be reused for several cycles.

The heating of protein preparations of mesophilic organism (e.g., E. coli) produces the obliteration of all soluble multimeric proteins from this organism. In this way, if a multimeric enzyme from a thermophilic microorganism is expressed in these mesophilic hosts, the only large protein remaining soluble in the preparation after heating is the thermophilic enzyme. These large proteins may be then selectively adsorbed on lowly activated anionic exchangers, enabling their full purification in just these two simple steps. This strategy has been applied to the purification of an R-galactosidase and a -galactosidase from Thermus sp. strain T2, both expressed in E. coli, achieving the almost full purification of both enzymes in only these two simple steps. This very simple strategy seems to be of general applicability to the purification of any thermophilic multimeric enzyme expressed in a mesophilic host.

A 3D cross-ply micromechanical model is used to analyse the thermomechanical behaviour of copper matrix composite reinforced with SiC fibres, when subjected to cyclic loadings at high temperature. The copper matrix composite is reinforced with 45% fibre volume fraction. A cohesive model is employed to capture the influence of the debonding interface in the composite, during the consolidation and subsequent thermal and mechanical loading.

This paper described that glyoxyl activated supports are only able to quantitatively immobilize proteins at alkaline pH values and using highly activated supports (otherwise, immobilization is negligible). Furthermore, the immobilization rate depends on the surface density of glyoxyl groups and not on the total concentration of groups in the suspension. Moreover, the temperature presented a dramatic effect on enzyme immobilization rate and different enzymes become immobilized at very different rates. Finally, it was not possible to detect immobilization of mono aminated compounds at neither alkaline nor neutral pH values. In fact, these supports may be used to purify enzymes due to these great differences in immobilization rate (e.g., glutamate racemase). These special features are not shared with other supports usually utilized for covalent immobilization of enzymes (e.g., glutaraldehyde, cyanogen bromide supports) that are able to immobilize proteins by just one very stable bond and suggesting an unique mechanism for glyoxyl agarose supports.

Lipase from Burkholderia cepacia (formerly Pseudomonas fluorescens) (BCL) was previously immobilized on glyoxyl-agarose and the active site was blocked after incubation with diethyl-p-nitrophenylphosphate, obtaining a new "glyoxyl-BCL * " matrix to adsorbe lipases. Then, the soluble lipase was offered to this matrix at very low ionic strength. Under these conditions, the lipase was selectively adsorbed on this new matrix. This lipase-lipase interaction could be neglected with the use of Triton X-100 as detergent. In addition, the close contact between the adsorbed lipase and the immobilized lipase permitted to alter the catalytic and functional properties of this lipase. For example, the enantioselectivity of the BCL adsorbed on glyoxyl-BCL * varied its E value from 10 at pH 7 and 25 • C up to >100 at pH 5 and 25 • C in the hydrolytic resolution of (±)-2-hydroxy-4-phenylbutyric acid ethyl ester. These results can open new routes for the modulation of lipase properties.

The interaction of two large beta-galactosidases (from Escherichia coli and from Thermus sp.) with tailor-made anion exchangers was studied. Using lowly activated supports (e.g., containing 2-3 mol of ionised groups per wet gram of support), large proteins selectively adsorbed and easily desorbed (e.g., using 200 mM of NaCl), giving highly purified proteins. However, these supports cannot be used to immobilize the enzymes for industrial use, because the weak adsorption.

PEI coated supports are proposed in this manuscript as a matrix that may permit a new concept in chromatography. Standard supports have their active groups on a "plane" surface, and permit the interaction with an area accounting for not more than 15-20% of the protein surface. The PEI coated supports permitted the interaction of the protein and a polymeric bead, where the protein may penetrate, permitting the interaction with a larger percentage of the protein surface. Here, we present that PEI coated supports are able to more strongly adsorb proteins that are adsorbed on conventional supports and to adsorb proteins that are not adsorbed on these supports. Moreover, more interestingly, the relative adsorption strength was found to be quite different when using PEI coated supports or conventional supports, because the distribution of charged groups in the protein surface may affect very distinctly to the adsorption when using each of these supports. Thus, some proteins that are very strongly adsorbed on conventional supports (having a small area with many anionic groups) were not among the most strongly adsorbed proteins on PEI coated supports, while some proteins that could not be adsorbed on conventional supports (that is, not having a very rich area in anionic groups) become very strongly adsorbed on PEI supports (very likely because they have dispersed around the surface many anionic groups). That is, the desorption pattern from both kind of supports is very different. We presented the results obtained in the fractioning of crude proteins extract from E. coli and whey protein concentrate. The sequential use of DEAE-agarose and PEI permit to improve the purity of the proteins adsorbed on PEI coated supports by more than a 10-fold factor and their further desorption may give only two to three bands in many instances.

The ␤-galactosidase from Thermus sp. T2 (Htag-BgaA) is competitively inhibited by galactose (3.1 mM) and non-competitively inhibited by glucose (49.9 mM). These inhibitions were strongly reduced by immobilization on heterofunctional epoxy Sepabeads (boronate-epoxy-Sepabeads and chelate-epoxy-Sepabeads). The immobilized preparations displayed increased competitive inhibition constants (K i ) of galactose (boronate-epoxy-Sepabeads, 12.5 mM and chelate-epoxy-Sepabeads, 11.7 mM), whilst the enzyme K M (lactose) only doubled its value. A significant increment of the non-competitive constant was also found (by around a two-fold factor).

The immobilization of the enzyme ␤-galactosidase from Thermus sp. T2 was performed via ionic adsorption onto two different supports: a new anionic exchanger resin, based on the coating of Sepabeads internal surfaces with polyethylenimine (PEI) polymers (M w 25,000), and conventional DEAE-agarose. Immobilization proceeded very rapidly in both cases, but the adsorption strength was much higher in the case of PEI-Sepabeads than in DEAE-supports at both pH 5 and 7 (e.g. at pH 7 and 0.4 M NaCl, less than 5% of enzyme was eluted from PEI-support while more than 70% protein was eluted from DEAE-agarose). Interestingly, the PEI-derivatives remained almost fully active at pH 5 and 7 after several weeks of incubation at 50 • C, conditions that allows the hydrolysis of lactose in milk coupled with the antimicrobial treatment usually performed.

The gene of a thermostable alpha-galactosidase, aglA, from Thermus sp. strain T2 was sequenced, cloned and overexpressed in Escherichia coli (strain MC 2508. The purified enzyme proved to be quite thermostable, retaining high levels of activity even after incubation at 70°C. The optimal T was 65°C at pH 7. The highest activity was achieved at acid pH values, although good activity could be obtained in a broad pH range. Enzyme stability depends on the enzyme concentration at alkaline pH values, suggesting that under these conditions the subunit dissociation may be the first step in the inactivation of the enzyme. However, at pH 5 the enzyme stability becomes independent of the enzyme concentration. The enzyme also exhibited a quite broad specificity, although it shows the best activity with alpha derivatives of galactose, and was able to recognize very different substrates (alpha derivatives of mannose, xylose and maltose, and even beta galactose derivatives). There was no detectable activity against glucose derivatives.