Vytas K. Švedas

Guidelines for reporting of biocatalytic reactions.

Enzymes and whole cells are being increasingly applied in research and industry, but the adoption of biocatalysis relies strongly on useful scientific literature. Unfortunately, too many published papers lack essential information needed to reproduce and understand the results. Here, members of the scientific committee of the European Federation of Biotechnology Section on Applied Biocatalysis (ESAB) provide practical guidelines for reporting experiments. The document embraces the recommendations of the STRENDA initiative (Standards for Reporting Enzymology Data) in the context of pure enzymology and provides further guidelines and explanations on topics of crucial relevance for biocatalysis. In particular, guidelines are given on issues such as the selectivity, specificity, productivity and stability of biocatalysts, as well as on methodological problems related to reactions in multiphase systems. We believe that adoption and use of these guidelines could greatly increase the value and impact of published work in biocatalysis, and hence promote the further growth of applications.

Biocatalytic approach to enantiomerically pure β-amino acids

β-Aryl-β-amino acids were prepared in good chemical yield and high enantiomeric purity (>95% ee) via penicillin acylase-catalyzed hydrolysis of the corresponding N-phenylacetyl derivatives. The (R)-enantiomers were the fast-reacting isomers in all cases studied. The biocatalytic procedure described employs very simple set of reactions using inexpensive commercially available chemicals and enzyme, and could be easily scaled up.

Substrate specificity of penicillin amidase from E. coli

1. The kinetic parameters of 12 substrates of penicillin amidase (penicillin amidohydrolase, EC 3.5.1.11) from E. coli have been determined. Most of the penicillin amidase amide substrates containing a phenylacetyl group in the acyl moiety have been shown to have similar catalytic constants of 50 s-1. Substitution of the phenylacetyl group b 2-thienylacetyl group (cephalothin, cephaloridine) having a similar structure leads to a slight decrease in kcat. 2. Nonspecific penicillin amidase substrates, which contain a free amino group in their acyl moiety, are characterized by a strong dependence of kcat, on the structure of the leaving group with Km being constant. To investigate the free amino group influence on the reaction kinetics, pH-dependences of kcat/Km of enzymatic hydrolysis of phenylacetic and D-(-)-alpha-aminophenylacetic acid p-nitroanilides have been studied. It has been shown that enzyme binds the deprotonated form of the substrate only. 3. Under thermodynamically favourable conditions for the synthesis of beta-lactam antibiotics (at low pH), a concentration of the deprotonated substrate form is very low, and the reaction proceeds in the bimolecular regime. The value of the second-order rate constant for the substrate having a free amino group is small even at pH 7.5, and sharply decreases as does the pH. Hence, despite the favourable thermodynamic conditions for the production of all beta-lactam antibiotics, low reaction rate is the basic hindrance for enzymatic synthesis of penicillins and cephalosporins having a free amino group in the acyl moiety.

Enzymatic synthesis of β-lactam antibiotics: A thermodynamic background

The equilibrium constants and the respective standard Gibbs energy changes for hydrolysis of some β-lactam antibiotics have been determined. Native and immobilized penicillin amidase (EC 3.5.1.11) from Escherichia coli has been used as a catalyst. The values of standard Gibbs energy changes corresponding to the pH-independent product of equilibrium concentrations (ΔG0c = − RT ln Kc) have been calculated. The differences in the structure of the antibiotics nucleus hardly ever affect the value of the pH-independent component of the standard Gibbs energy change (ΔG0c) and value of apparent standard Gibbs energy change at a fixed pH (ΔG0′c). At the same time, the value of ΔG0c is more sensitive to the structure of the acyl moiety of the antibiotic; when ampicillin is used instead of benzylpenicillin, ΔG0c increases by ∼6.3 kJ mol−1 (1.5 kcal mol−1). pH-dependences of the apparent standard Gibbs energy changes for hydrolysis of β-lactam antibiotics have been calculated. The pH-dependences of ΔG0′c for hydrolysis of all β-lactam antibiotics have a similar pattern. The thermodynamic pH optimum of the synthesis of these compounds is in the acid pH range (pH < 5.0). The breakage of the β-lactam ring leads to a sharp decrease in the ΔG0′c value and a change in the pattern of the pH-dependence. For example, at pH 5.0 ΔG0′c decreases from 14.4 kJ mol−1 for benzylpenicillin to −1.45 kJ mol−1 for benzylpenicilloic acid. The reason for these changes is mainly a considerable increase in the pK of the amino group of the nucleus of the antibiotic and, as a consequence, a decrease in the component of standard Gibbs energy change, corresponding to the ionization of the system. The thermodynamic potentials of the enzymatic synthesis of semisynthetic penicillins and cephalosporins on the basis of both free acids and their derivatives (N-acylated amino acids, esters) are discussed. It is shown that with esters of the acids, a high yield of the antibiotic can, in principle, be achieved at higher pH values.

Penicillin acylase-catalyzed resolution of amines in aqueous organic solvents

Abstract Penicillin acylase from Alcaligenes faecalis catalyzes the enantioselective acylation of amines with phenylacetamide in a kinetically controlled reaction in water at pH 11. Addition of cosolvent to the reaction mixture significantly improved the enantioselectivity in most cases. Penicillin acylase from E. coli also catalyzed the phenylacetylation of amines, but an order of magnitude less efficiently than with the enzyme of A. faecalis . Amine resolution via kinetically controlled acylation competes effectively with hydrolysis of N -acylated compounds and constitutes a synthetically useful alternative to existing lipase-based methods.

Biocatalytic resolution of β-fluoroalkyl-β-amino acids

Abstract N -Phenylacetyl derivatives of β-fluoroalkyl-β-alanines 6 were synthesized and biocatalytically resolved to the corresponding enantiopure β-amino acids 7,9 with the aid of penicillin acylase (EC 3.5.1.11) from Escherichia coli . In substrates 6 the enantioselectivity of the biocatalytic process was practically uninfluenced by the nature of the fluoroalkyl chain. Thus, β-fluoroalkyl-β-alanines 7,9 bearing short (R = CF 3 , CHF 2 ) or long [C 3 F 7 , H(CF 2 )4] chains were prepared in high enantiomeric parity. The ( R )-enantiomer was the fast-reacting enantiomer in all cases.

Chemo-enzymatic approach to the synthesis of each of the four isomers of α-alkyl-β-fluoroalkyl-substituted β-amino acids

Abstract Starting from easily available ethyl 2-methyl-4,4,4-trifluoroacetoacetate and benzylamine each of the four stereoisomers of α-methyl-β-trifluoromethyl-β-alanine have been synthesized in optically pure form via stereocontrolled chemo-enzymatic procedure including diastereoselective base-catalyzed [1,3]-proton shift reaction and enantioselective penicillin acylase-catalyzed resolution.

KINETIC STUDY OF PENICILLIN ACYLASE FROM ALCALIGENES FAECALIS

Penicillin acylase from Alcaligenes faecalis has a very high affinity for both natural (benzylpenicillin, K m=0.0042 mM) and colorimetric (6‐nitro‐3‐phenylacetamidobenzoic acid, K m=0.0045 mM) substrates as well as the product of their hydrolysis, phenylacetic acid (K i=0.016 mM). The enzyme is partially inhibited at high benzylpenicillin concentrations but the triple SES complex formed still retains 43% of the maximal catalytic activity; the affinity of benzylpenicillin for the second substrate molecule binding site is much lower (K S′=54 mM) than for the first one. Phenylmethylsulfonyl fluoride was shown to be a very effective irreversible inhibitor, completely inactivating the penicillin acylase from A. faecalis in a few minutes at micromolar concentrations; this compound was used for enzyme active site titration. The absolute values of the determined kinetic parameters for enzymatic hydrolysis of 6‐nitro‐3‐phenylacetamidobenzoic acid (k cat=95 s−1 and k cat/K m=2.1×10−7 M−1 s−1) and benzylpenicillin (k cat=54 s−1 and k cat/K m=1.3×10−7 M−1 s−1) by penicillin acylase from A. faecalis were shown to be highest of all the enzymes of this family that have so far been studied.

Highly efficient and enantioselective enzymatic acylation of amines in aqueous medium

Abstract A new strategy based on the unique catalytic properties, stability and enantioselectivity of the relatively unknown penicillin acylase from Alcaligenes faecalis has been developed for the effective and enantioselective acylation of amines in aqueous medium. In contrast to lipase-catalyzed acylations in organic solvents, the penicillin acylase-catalyzed acylation of amines in aqueous solution is a rapid and chemoselective process leading to a product which can subsequently be deacylated by the same enzyme, imposing secondary enantiocontrol and leading to effective resolution.

Quantitative characterization of the nucleophile reactivity in penicillin acylase-catalyzed acyl transfer reactions.

Nucleophile reactivity of two most known nuclei of penicillins and cephalosporins, 6-aminopenicillanic (6-APA) and 7-aminodesacetoxycephalosporanic (7-ADCA) acids, was quantitatively characterized. In penicillin acylase (PA)-catalyzed acyl transfer reactions the relative reactivity of the added nucleophile compared to the water (i.e. nucleophile reactivity) is defined by two complex kinetic parameters beta(0) and gamma, and depends on the nucleophile concentration. In turn, parameters beta(0) and gamma were shown to be dependent on the structure of both reactants involved: nucleophile and acyl donor. Analysis of the kinetic scheme revealed that nucleophile reactivity is one of a few key parameters controlling efficiency of PA-catalyzed acyl transfer to the added nucleophile in an aqueous medium. Computation of the maximum nucleophile conversion to the product using determined nucleophile reactivity parameters in the synthesis of three different antibiotics, ampicillin, amoxicillin and cephalexin, showed good correlation with the results of corresponding synthetic experiments. Suggested approach can be extended to the quantitative description and optimization of PA-catalyzed acyl transfer reactions in a wide range of experimental conditions.