Processing and functional display of the 86 kDa heterodimeric penicillin G acylase on the surface of phage fd.

The large heterodimeric penicillin G acylase from Alcaligenes faecalis was displayed on the surface of phage fd. We fused the coding sequence (alpha subunit-internal peptide-beta subunit) to the gene of a phage coat protein. A modified g3p signal sequence was used to direct the polypeptide to the periplasm. Here we show that a heterodimeric enzyme can be expressed as a fusion protein that matures to an active biocatalyst connected to the coat protein of phage fd, resulting in a phage to which the beta-subunit is covalently linked and the alpha-subunit is non-covalently attached. The enzyme can be displayed either fused to the minor coat protein g3p or fused to the major coat protein g8p. In both cases the penicillin G acylase on the phage has the same Michaelis constant as its freely soluble counterpart, indicating a proper folding and catalytic activity of the displayed enzyme. The display of the heterodimer on phage not only allows its further use in protein engineering but also offers the possibility of applying this technology for the excretion of the enzyme into the extracellular medium, facilitating purification of the protein. With the example of penicillin acylase the upper limit for a protein to become functionally displayed by phage fd has been further explored. Polyvalent display was not observed despite the use of genetic constructs designed for this aim. These results are discussed in relation to the pore size being formed by the g4p multimer.

Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis.

Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge.

Description of enzyme kinetics in reversed micelles. 1. Theory.

In the literature measurements of kinetic data of enzymes in reversed micelles have been interpreted in two ways. In the first, all enzyme parameters are expressed with respect to the total volume of the reversed micellar solution. In the second, the enzymatic conversion is related only to the fraction of the volume consisting of aqueous solution (pseudophase model). In this paper equations are derived describing the rate of an enzymatic reaction for three different kinds of enzymes: enzymes obeying Michaelis-Menten kinetics, enzymes following a ping-pong bi-bi mechanism and enzymes which convert substrates according to an ordered mechanism. In deriving these equations, a distinction is made between intermicellar exchange reactions of substrate(s) and product(s) and the enzymatic reaction which takes place in the waterpool of a reversed micelle. In the description, all intrinsic rate constants of the enzyme are assumed to be independent of its environment. The rate equations show that the presence and efficiency of the intermicellar exchange reaction, which supplies the enzyme with substrate and removes product, can affect the rate of an enzymatic reaction under common experimental conditions. Whereas kinetic parameters derived from double-reciprocal plots often seem to be affected by enclosure in reversed micelles, these apparent deviations from kinetics in aqueous media can be explained by the model presented here as arising from exchange phenomena. Neither the experimentally determined maximum enzyme velocity, vmax, nor the Michaelis constants are affected by the incorporation of the enzyme in reversed micelles. The deviations of kinetic parameters from the aqueous values are shown to depend strongly on the concentration of reversed micelles, the intermicellar exchange rate and the volume fraction of water, a dependence in agreement with findings reported in the literature.

Enzyme kinetics in reversed micelles. 2. Behaviour of enoate reductase.

Enoate reductase (EC 1.3.1.31) can stereospecifically reduce a variety of alpha,beta-unsaturated carboxylates. Its use was extended to apolar media by incorporating the enzyme into a reversed micellar medium. The kinetics of the enzyme in such a medium have been investigated using 2-methylbutenoic acid as substrate and NADH as a cofactor and compared with the reaction rates in aqueous solution. In aqueous solution the enzyme obeys a ping pong mechanism [Bühler et al. (1982) Hoppe-Seyler's Z. Physiol. Chem 363, 609-625]. In 50 mM Hepes pH = 7.0 with ionic strength of 0.05 M the Michaelis constants for NADH and 2-methylbutenoic acid are 20 microM and 6.0 mM respectively. In reversed micelles the kinetics of the reaction (Michaelis constant, maximum velocity as well as inhibitory effects) were markedly different. The rate of the enzymatic reaction of enoate reductase was studied using various concentrations of 2-methylbutenoic acid and various NADH concentrations. In reversed micelles composed of the anionic detergent sodium di(ethylhexyl)sulphosuccinate, the enzymatic reaction deviates substantially from the values in aqueous solution. Using our model (see preceding paper in this issue of the journal), all kinetics could be explained as evolving from enclosure in reversed micelles without any change in the intrinsic rate parameters of the enzyme. So the enzyme itself is unaffected by incorporation in reversed micelles, but the rate of intermicellar exchange as well as the microheterogeneity of the medium, resulting in very high local concentrations of the substrate, are the most important factors altering the reaction pattern. The effect of the composition of the reversed micellar medium was also investigated using either a nonionic or a cationic surfactant. In these solutions too, exchange and microheterogeneity of the medium proved to be the most important parameters influencing the enzymatic reaction. In all reversed micellar solutions inhibition by the enoate was observed at an overall concentration of 0.5-5 mM, implying that a concentration of substrate equal to the Km value in aqueous solution may already cause inhibition in reversed micelles. At this level no inhibition by NADH was observed. The microheterogeneity of the medium also explains this inhibition of the enzyme at relatively low 2-methylbutenoic acid concentrations.

Enzyme kinetics in reversed micelles. 3. Behaviour of 20 beta-hydroxysteroid dehydrogenase.

The kinetic parameters of 20 beta-hydroxysteroid dehydrogenase were determined in aqueous solutions and in reversed micellar media composed with either an anionic, a cationic or a nonionic surfactant, at low and at high ionic strength. The velocity data were analysed in two ways: first by extrapolation to infinite concentrations of both substrates to determine 'apparent' Michaelis constants and V values, and secondly by comparison to reaction rates calculated using the model presented (see first of this series of papers in this issue of the journal). Data analysis according to the first method reveals some differences in the kinetic parameters in reversed micelles as compared to those in aqueous solution, though the kinetic parameters of the enzyme seem not to be much affected by enclosure in reversed micelles. It is shown that the changes that do occur are not caused by a shift of the intramicellar pH or by electrostatic interactions between the enzyme and the surfactant head groups. Interpretation of the data using the second method assumes that the enzyme is not affected by the enclosure in reversed micelles, and that deviations with respect to the aqueous parameters are caused by exchange phenomena between distinct aqueous droplets in the organic phase and by a high effective intramicellar substrate concentration. This model is able to predict reaction rates that agree rather well with experimentally determined rates and explains why the enzyme mechanism in reversed micelles is, at all progesterone concentrations used, the same as observed at high progesterone concentrations in aqueous solution. Furthermore it clarifies the occurrence of substrate inhibition in sodium-di(ethylhexyl)sulphosuccinate-reversed micelles and the observed low activity in Triton-reversed micelles, as arising from the high partition coefficient of progesterone and the slow rate of diffusion of progesterone into the reversed micelles. From these results, and those reported for enoate reductase (see preceding paper in this issue of the journal) it can be concluded that the theory presented before (see first of this series of papers in this issue of the journal) offers a good explanation for the observed kinetic behaviour in reversed micelles, and emphasizes the importance of exchange processes between micelles.