Marco Terreni

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.

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.

Use of aqueous two-phase systems for in situ extraction of water soluble antibiotics during their synthesis by enzymes immobilized on porous supports.

Yields of kinetically controlled synthesis of antibiotics catalyzed by penicillin G acylase from Escherichia coli (PGA) have been greatly increased by continuous extraction of water soluble products (cephalexin) away from the surroundings of the enzyme. In this way its very rapid enzymatic hydrolysis has been avoided. Enzymes covalently immobilized inside porous supports acting in aqueous two-phase systems have been used to achieve such improvements of synthetic yields. Before the reaction is started, the porous structure of the biocatalyst can be washed and filled with one selected phase. In this way, when the pre-equilibrated biocatalyst is mixed with the second phase (where the reaction product will be extracted), the immobilized enzyme remains in the first selected phase in spite of its possibly different natural trend. Partition coefficients (K) of cephalexin in very different aqueous two-phase systems were firstly evaluated. High K values were obtained under drastic conditions. The best K value for cephalexin (23) was found in 100% PEG 600-3 M ammonium sulfate where cephalexin was extracted to the PEG phase. Pre-incubation of immobilized PGA derivatives in ammonium sulfate and further suspension with 100% PEG 600 allowed us to obtain a 90% synthetic yield of cephalexin from 150 mM phenylglycine methyl ester and 100 mM 7-amino desacetoxicephalosporanic acid (7-ADCA). In this reaction system, the immobilized enzyme remains in the ammonium sulfate phase and hydrolysis of the antibiotic becomes suppressed because of its continuous extraction to the PEG phase. On the contrary, synthetic yields of a similar process carried out in monophasic systems were much lower (55%) because of a rapid enzymatic hydrolysis of cephalexin.

Different phyllosilicates as supports for lipase immobilisation

The aim of this work was to determine the enzymatic activities resulting from the adsorption of Rhizomucor miehei lipase (RML) and Candida cylindracea lipase (CCL) onto three different phyllosilicates (sepiolite, palygorskite and montmorillonite), comparing the resultant activities with those obtained following similar immobilisation technique on a widely used resin (Duolite A-568). Due to the different adsorption mechanisms produced, different derivatives with higher hydrolytic activities can be obtained. Comparing the clays tested, the results showed that, in comparison with the laminar silicate (montmorillonite sample) and Duolite A-568 (spherical particles), fibrous materials (palygorskite and sepiolite) resulted in derivatives with higher hydrolytic activities in the hydrolysis of different ethyl esters. Moreover, according to the data obtained with the electrophoresis, the selectivity of immobilisation for RML in the case of fibrous silicates was optimal. As a conclusion, and according to the activities and selectivities measured, at least two out of the four studied materials (sepiolite and palygorskite) would be useful as supports for immobilisation for proteins of relatively low molecular weight (such as RML) for further use in biotransformations, while for C. cylindracea the immobilisation onto duolite rendered a derivative specially active in the hydrolysis of ethyl formiate (esterasic activity).

Evaluation of different enzymes as catalysts for the production of β-lactam antibiotics following a kinetically controlled strategy

Abstract Several β-lactam acylases produced by different microorganisms ( Escherichia coli , Kluyvera citrophila , Acetobacter turbidans , and Bacillus megaterium ) have been evaluated as catalysts for the syntheses of relevant β-lactam antibiotics (ampicillin, cephalexin, and cefamandole). These enzymes displayed very different synthetic properties showing large differences in synthetic yields (by a 4- to 5-fold factor) depending on the antibiotic and the enzyme. The enzyme from A. turbidans presented the best properties for the synthesis of ampicillin, which is a low activity in the hydrolysis of the antibiotic and a high specificity for the transformation of the ester into antibiotic. Although this enzyme was able to transform approximately 80% of phenylglycine methyl ester into ampicillin, it was unsuitable for the synthesis of cephalexin and cefamandole. In fact, all of the enzymes showed significant hydrolysis rates of the antibiotics compared to the synthetic activity, although the enzyme from E. coli exhibited the highest specificity for the transformation of esters into these antibiotics. To prevent the hydrolysis of the antibiotic, a two-phase aqueous system was used to extract the antibiotic from the enzyme environment. In this way, high synthetic yields could be obtained, e.g. 80% of phenylglycine methyl ester was transformed into cephalexin using the enzyme from E. coli .

A criterion for the selection of monophasic solvents for enzymatic synthesis

Abstract Penicillin acylase can be used for the synthesis of β-lactamic antibiotics which have a wide range of applications; however, the utility of this synthetic route is often limited by the necessity of using organic solvents which have both an inhibitory effect on the enzyme activity and a deleterious effect on the enzyme stability. The first limitation can be overcome by careful selection of the organic solvent so as to ensure that high synthetic yields are obtained without excessively reducing the activity of the enzyme derivative, and the second by using enzymes previously stabilized. In this paper, the synthetic and hydrolytic behavior of stabilized derivatives of penicillin acylase has been tested in various water-cosolvent mixtures. The overall performance of the enzyme in the synthetic mode is a combination of two effects: (i) the inhibitory effect of the solvent on the enzyme and (ii) the desirable increase in the nonionized form of the substrate in the presence of solvent. The ratio of the enzyme activity in its synthetic mode to its hydrolytic mode was found to be a useful guide for the selection of a suitable solvent.

Regio-selective deprotection of peracetylated sugars via lipase hydrolysis

Abstract Purified lipases (via interfacial activation on hydrophobic supports) from different microbial extracts have been evaluated in the regio-selective hydrolysis of peracetylated sugars (peracetylated glucose, ribose and sucrose). Among the enzymes tested, lipases from Candida rugosa (CRL) and from Pseudomonas fluorescens (PFL) exhibited the best properties in these reactions. Then, we have prepared two different immobilized lipase preparations obtained by interfacial activation on hydrophobic supports or by covalent attachment on glutaraldehyde agarose. Interfacially activated lipases exhibited a higher activity than covalently attached enzymes (even by a 100-fold factor), giving the higher yields of mono deacetylated sugars (in some instances by more than a threefold factor) in short reaction times. In the hydrolysis of 1,2,3,5-tetra-O-acetyl-β- d -ribofuranose catalyzed by PFL adsorbed on octyl agarosa, hydrolyzed mainly the 3 position (30% of yield) while the CRL gave the hydrolysis only in position 5 (about 50% of yield). Depending on the enzyme immobilized preparation, we have been able also to obtain selective hydrolysis of 1,2,3,4,6-penta-O-acetyl-α/β- d -glucopyranose obtaining a free hydroxyl group in position 1, 4 or 6. Moreover, selective hydrolysis in the 4′ position of peracetylated sucrose was achieved when the hydrolysis is performed with CRL immobilized on octyl-agarose (yield was 77%).

Biocatalyst engineering exerts a dramatic effect on selectivity of hydrolysis catalyzed by immobilized lipases in aqueous medium

It has been found that enantioselectivity of lipases is strongly modified when their immobilization is performed by involving different areas of the enzyme surface, by promoting a different degree of multipoint covalent immobilization or by creating different environments surrounding different enzyme areas. Moreover, selectivity of some immobilized enzyme molecules was much more modulated by the experimental conditions than other derivatives. Thus, some immobilized derivatives of Candida rugosa (CRL) and C. antarctica-B (CABL) lipases are hardly enantioselective in the hydrolysis of chiral esters of (R,S)-mandelic acid under standard conditions (pH 7.0 and 25°C) (E < 2). However, other derivatives of the same enzymes exhibited a very good enantioselectivity under nonstandard conditions. For example. CRL adsorbed on PEI-coated supports showed a very high enantio-preference towards S-isomer ( E = 200) at pH 5. On the other hand, CABL adsorbed on octyl-agarose showed an interesting enantio-preference towards the R-isomer ( E = 25) at pH 5 and 4°C. These biotransformations are catalyzed by isolated lipase molecules acting on fully soluble substrates and in the absence of interfacial activation against external hydrophobic interfaces. Under these conditions, lipase catalysis may be associated to important conformational changes that can be strongly modulated via biocatalyst and biotransformation engineering. In this way, selective biotransformations catalyzed by immobilized lipases in macro-aqueous systems can be easily modulated by designing different immobilized derivatives and reaction conditions.

Pen G acylase catalyzed resolution of phenylacetate esters of secondary alcohols

Abstract Penicillin G acylase from E. coli (E.C. 3.5.1.11.) immobilized on Eupergit C is used for the kinetic resolution of phenyl acetate esters of secondary alcohols of pharmaceutical interest.

Different Properties of the Lipases Contained in Porcine Pancreatic Lipase Extracts as Enantioselective Biocatalysts

The porcine pancreatic lipase (PPL) extracts contain a mixture of several lipases. Their fractioning was performed by sequential adsorption via interfacial activation on supports with different hydrophobicity. A protein of 25 KDa was preferentially adsorbed on octyl‐Sepharose, another protein of 33 kDa was mainly adsorbed on octadecyl‐Sepabeads support, and the PPL was mainly adsorbed on the support bearing phenyl groups. The different immobilized preparations showed different properties and different response due to change in the experimental conditions. Thus, in the hydrolysis of (±)‐2‐hydroxy‐4‐phenylbutyric acid ethyl ester [(±)‐1] to produce the corresponding acid [2], the octyl‐25KDa preparation showed the best enantioselectivity ( E) value ( E = 7) at pH 5 and 25 °C, whereas the phenyl‐PPL was the most enantioselective ( E = 10) at pH 5, 4 °C, and 10% dioxane. Using different preparations at different pHs it was possible to resolve (±)‐2‐ O‐butyryl‐2‐phenylacetic acid [(±)‐3] with a high E value ( E > 100); for example, with octadecyl‐33 KDa enzyme at pH 8.