A self-immolative linker that releases thiols detects penicillin amidase and nitroreductase with high sensitivity via absorption spectroscopy.

This article reports the synthesis and characterization of a novel self-immolative linker, based on thiocarbonates, which releases a free thiol upon activation via enzymes. We demonstrate that thiocarbonate self-immolative linkers can be used to detect the enzymes penicillin G amidase (PGA) and nitroreductase (NTR) with high sensitivity using absorption spectroscopy. Paired with modern thiol amplification technology, the detection of PGA and NTR were achieved at concentrations of 160 nM and 52 nM respectively. In addition, the PGA probe was shown to be compatible with both biological thiols and enzymes present in cell lysates.

Integrative strategy to determine residual proteins in cefaclor produced by immobilized penicillin G acylase.

There is a growing trend in the pharmaceutical industry towards substituting conventional chemical synthesis routes of semi-synthetic ß-lactam antibiotics (SSBAs) through environmentally sustainable enzymatic processes. These have advantages such as cost reduction in terms of solvent and waste treatment and time saving owing to fewer reaction steps. Penicillin G acylase (PGA) is an industrially important enzyme that is mainly used to catalyze the synthesis of SSBAs. In this study, we established an integrative strategy using three different analytical methods for determining the PGA-associated residual protein content, which is a critical quality issue in the end product. Cefaclor was taken as representative example of SSBAs. High-performance liquid chromatography coupled with fluorescence detection (HPLC-FD) allowed the routine analysis of PGA residual proteins and other low molecular weight (MW) impurities with high detection specificity and sensitivity, comparable to those of the Bradford assay and microfluidic protein chip electrophoresis. However, these latter two methods were superior for quantitative and qualitative analysis, respectively, and should be regarded as necessary adjuncts to the HPLC-FD method. By combining the three methods, trace levels of residual proteins were detected in four (out of 13) cefaclor bulk samples from two different manufacturers, with a major protein MW of ∼63 kDa. This suggests that the higher MW PGA subunit tends to persist in the end product. The integrative determination strategy described here can be used to evaluate SSBA bulk samples and monitor the process of SSBA manufacturing by enzymatic methods, especially in terms of inter-batch consistency and process stability.

Activity-Fed Translation (AFT) Assay: A New High-Throughput Screening Strategy for Enzymes in Droplets.

There is an increasing demand for the development of sensitive enzymatic assays compatible with droplet-based microfluidics. Here we describe an original strategy, activity-fed translation (AFT), based on the coupling of enzymatic activity to in vitro translation of a fluorescent protein. We show that methionine release upon the hydrolysis of phenylacetylmethionine by penicillin acylase enabled in vitro expression of green fluorescent protein. An autocatalytic setup where both proteins are expressed makes the assay highly sensitive, as fluorescence was detected in droplets containing single PAC genes. Adding a PCR step in the droplets prior to the assay increased the sensitivity further. The strategy is potentially applicable for any activity that can be coupled to the production of an amino acid, and as the microdroplet volume is small the use of costly reagents such as in vitro expression mixtures is not limiting for high-throughput screening projects.

Chiral separations by capillary electrophoresis using proteins as chiral selectors.

Capillary electrophoresis (CE) methods using proteins as the chiral selectors have been developed for the separation of enantiomeric mixtures. For chiral separations in protein-based CE, two methods were utilized. One is affinity capillary electrochromatography (ACEC), and the other is affinity CE (ACE). This chapter deals with the advantages and disadvantages of ACEC and ACE. Furthermore, enantioseparations utilizing ACEC based on packed α(1)-acid glycoprotein-immobilized silica gels, immobilized avidin to fused silica capillaries and ACE based on penicillin G-acylase dissolved in the running buffer are described in detail.

New generation of amino coumarin methyl sulfonate-based fluorogenic substrates for amidase assays in droplet-based microfluidic applications.

Droplet-based microfluidics is a powerful tool for biology and chemistry as it allows the production and the manipulation of picoliter-size droplets acting as individual reactors. In this format, high-sensitivity assays are typically based on fluorescence, so fluorophore exchange between droplets must be avoided. Fluorogenic substrates based on the coumarin leaving group are widely used to measure a variety of enzymatic activities, but their application in droplet-based microfluidic systems is severely impaired by the fast transport of the fluorescent product between compartments. Here we report the synthesis of new amidase fluorogenic substrates based on 7-aminocoumarin-4-methanesulfonic acid (ACMS), a highly water-soluble dye, and their suitability for droplet-based microfluidics applications. Both substrate and product had the required spectral characteristics and remained confined in droplets from hours to days. As a model experiment, a phenylacetylated ACMS was synthesized and used as a fluorogenic substrate of Escherichia coli penicillin G acylase. Kinetic parameters (k(cat) and K(M)) measured in bulk and in droplets on-chip were very similar, demonstrating the suitability of this synthesis strategy to produce a variety of ACMS-based substrates for assaying amidase activities both in microtiter plate and droplet-based microfluidic formats.

A multicomponent reaction-diffusion model of a heterogeneously distributed immobilized enzyme.

A physical model was derived for the synthesis of the antibiotic cephalexin with an industrial immobilized penicillin G acylase, called Assemblase. In reactions catalyzed by Assemblase, less product and more by-product are formed in comparison with a free-enzyme catalyzed reaction. The model incorporates reaction with a heterogeneous enzyme distribution, electrostatically coupled transport, and pH-dependent dissociation behavior of reactants and is used to obtain insight in the complex interplay between these individual processes leading to the suboptimal conversion. The model was successfully validated with synthesis experiments for conditions ranging from heavily diffusion limited to hardly diffusion limited, including substrate concentrations from 50 to 600 mM, temperatures between 273 and 303 K, and pH values between 6 and 9. During the conversion of the substrates into cephalexin, severe pH gradients inside the biocatalytic particle, which were previously measured by others, were predicted. Physical insight in such intraparticle process dynamics may give important clues for future biocatalyst design. The modular construction of the model may also facilitate its use for other bioconversions with other biocatalysts.

Fast and efficient protein purification using membrane adsorber systems.

The purification of proteins from complex cell culture samples is an essential step in proteomic research. Traditional chromatographic methods often require several steps resulting in time consuming and costly procedures. In contrast, protein purification via membrane adsorbers offers the advantage of fast and gentle but still effective isolation. In this work, we present a new method for purification of proteins from crude cell extracts via membrane adsorber based devices. This isolation procedure utilises the membranes favourable pore structure allowing high flow rates without causing high back pressure. Therefore, shear stress to fragile structures is avoided. In addition, mass transfer takes place through convection rather than diffusion, thus allowing very rapid separation processes. Based on this membrane adsorber technology the separation of two model proteins, human serum albumin (HSA) and immungluboline G (IgG) is shown. The isolation of human growth hormone (hGH) from chinese hamster ovary (CHO) cell culture supernatant was performed using a cation exchange membrane. The isolation of the enzyme penicillin acylase from the crude Escherichia coli supernatant was achieved using an anion exchange spin column within one step at a considerable purity. In summary, the membrane adsorber devices have proven to be suitable tools for the purification of proteins from different complex cell culture samples.

Field-emission scanning electron microscopy analysis of morphology and enzyme distribution within an industrial biocatalytic particle.

Field-emission scanning electron microscopy (FESEM) was used in a technical feasibility study to obtain insight into the internal morphology and the intraparticle enzyme distribution of Assemblase, an industrial biocatalytic particle containing immobilized penicillin-G acylase. The results were compared with previous studies based on light and transmission electron microscopic techniques. The integrated FESEM approach yielded the same quantitative results as the microscopic techniques used previously. Given this technical equivalence, the integrated approach offers several advantages. First, the single preparation method and detection system avoids interpretation discrepancies between corresponding areas that were examined for different properties with different detection techniques in different samples. Second, the specimen size suitable for whole particle study is virtually unlimited, which simplifies sectioning and puts less stringent demands on the embedding technique. Furthermore, the sensitivity toward enzyme presence and distribution increases because the epitopes inside thick sections become available for labeling. Quick and unambiguous analysis of the relation between particle morphology and enzyme distribution is important because this information may be used in the future for the design of enzyme distributions in which the particle morphology can be used as a control parameter.

Enzyme distribution and matrix characteristics in biocatalytic particles.

In a study of Assemblase, an industrial immobilized penicillin-G acylase, various electron microscopic techniques were used to relate intra-particle enzyme heterogeneity with the morphological heterogeneity of the support material at various levels of detail. Transmission electron microscopy was used for the study of intra-particle penicillin-G acylase distribution in Assemblase particles of various sizes; it revealed an abrupt increase in enzyme loading at the particle surface (1.4-fold) and in the areas (designated halo's) surrounding internal macro-voids (7.7-fold). Cryogenic field-emission scanning electron microscopy related these abrupt local enzyme heterogeneities to local heterogeneity of the support material by revealing the presence of dense top layers surrounding both the particle exterior and the internal macro-voids. Furthermore, it showed a very distinct morphological appearance of the halo. Most probably, all these regions contained relatively more chitosan than gelatin (the polymers Assemblase was constructed of), which suggested local polymer demixing during particle production. A basic thermodynamic line of reasoning suggested that a difference in hydrophilicity between the two polymers induced local demixing. In the future, thermodynamic knowledge on such polymer interactions resulting in matrix heterogeneity may be used as a tool for biocatalyst design.

Novel approach to quantify immobilized-enzyme distributions.

The quantitative intraparticle enzyme distribution of Assemblase, an industrially employed polydisperse immobilized penicillin-G acylase, was measured. Because of strong autofluorescence of the carrier, the generally applied technique of confocal scanning microscopy could not be used; light microscopy was our method of choice. To do so, Assemblase particles of various sizes were sectioned, labeled with antibodies specifically against the enzyme, and analyzed light microscopically. Image analysis software was developed and used to determine the intraparticle enzyme distribution, which was found to be heterogeneous, with most enzyme located in the outer regions of the particles. Larger particles showed steeper gradients than smaller ones. A mathematical representation of the intraparticle profiles, based on in-stationary enzyme diffusion into the particles, was validated successfully for a broad range of particle sizes using data for volume-averaged particle size and enzyme loading. The enzyme gradients determined in this work will be used as input for a physical model that quantitatively describes the complex behavior of Assemblase. Such a physical model will lead to identification of the current bottlenecks in Assemblase and can serve as a starting point for the design of improved biocatalysts that also may be based on intelligent use of enzyme gradients.

Evidence for the involvement of arginyl residue at the active site of penicillin G acylase from Kluyvera citrophila.

Penicillin G acylase (PGA) is used for the commercial production of semi-synthetic penicillins. It hydrolyses the amide bond in penicillin producing 6-aminopenicillanic acid and phenylacetate. 6-Aminopenicillanic acid, having the beta-lactam nucleus, is the parent compound for all semi-synthetic penicillins. Penicillin G acylase from Kluyvera citrophila was purified and chemically modified to identify the role of arginine in catalysis. Modification with 20 mM phenylglyoxal and 50 mM 2,3-butanedione resulted in 82% and 78% inactivation, respectively. Inactivation was prevented by protection with benzylpenicillin or phenylacetate at 50 mM. The reaction followed psuedo-first order kinetics and the inactivation kinetics (V(max), K(m), and k(cat)) of native and modified enzyme indicates the essentiality of arginyl residue in catalysis.

Penicillin G acylase-based stationary phases: analytical applications.

A review of Penicillin G Acylase (PGA)-based stationary phases is given, focusing on immobilisation methods, selection of immobilisation material and applications in chiral liquid chromatography. Two immobilization methods, namely "in situ" and "in batch" techniques, are described for the immobilisation of PGA on silica supports. Microparticulate and monolithic silica, both functionalized with aminopropyl- and epoxy-groups, were used in the development of the PGA immobilised enzyme reactor (IMER). The best results, in terms of PGA immobilised amount and enzyme activity, were obtained with the "in situ" immobilisation on epoxy monolithic silica. The use of PGA columns as enzyme reactors for the preparation of 6-APA and for the production of enantiomeric pure drugs in a one-step reaction in described. The review also covers the application of PGA-columns as chiral stationary phases for the separation of acidic enantiomers. An on-line chromatographic system based on the PGA-IMER combined with a switching valve to an analytical column is also described as a highly efficient tool to study the enantioselective hydrolyses properties of PGA. Finally a molecular modelling study is reported with the aim to give more insights into PGA-substrates interactions and to expand the application of these stationary phases as a chiral biocatalysts for pharmaceutical processes.

[Selection of A strain producing beta-lactam antibiotics acylases].

A series of substrates analogues containing the same or similar side chain of its substrate have been synthesized and applied to screen cephalosporin acylase producers from a mass of microorganisms. The deacylation products of these analogues can be detected conveniently and used as screening indicators for the cephalosporin-acylase-producing-microorganism. Six strains possessing deacylation activity have been screened out with these substrate analogs. Among them, strain ZH0650 can simultaneously hydrolyze GL-7ACA, NIPAB and other analogues including AD-NABA. Further investigation on this strain confirmed that it could produce at least three acylases, ADNABA acylase, penicillin G acylase and cephalosporin acylase, which were characterized by bioassay with multiple substrate analogues. This is the first report that three different acylases were produced by one strain.

Active site titration as a tool for the evaluation of immobilization procedures of penicillin acylase.

Native and immobilized preparations of penicillin acylase from Escherichia coli and Alcaligenes faecalis were studied using an active site titration technique. Knowledge of the number of active sites allowed the calculation of the average turnover rate of the enzyme in the various preparations and allowed us to quantify the contribution of irreversible inactivation of the enzyme to the loss of catalytic activity during the immobilization procedure. In most cases a loss of active sites as well as a decrease of catalytic activity per active site (turnover rate) was observed upon immobilization. Immobilization techniques affected the enzymes differently. The effect of increased loading of penicillin acylase on the average turnover rate was determined by active site titration to assess diffusion limitations in the carrier.

Evaluation of the performance of immobilized penicillin G acylase using active-site titration.

Penicillin G acylase from Escherichia coli was immobilized on Eupergit C with different enzyme loading. The activity of the immobilized preparations was assayed in the hydrolysis of penicillin G and was found to be much lower than would be expected on the basis of the residual enzyme activity in the immobilization supernatant. Active-site titration demonstrated that the immobilized enzyme molecules on average had turnover rates much lower than that of the dissolved enzyme. This was attributed to diffusion limitations of substrate and product inhibition. Indeed, when the immobilized preparations were crushed, the activity increased from 587 U g-1 to up to 974 U g-1. The immobilized preparations exhibited up to 15% lower turnover rates than the dissolved enzyme in cephalexin synthesis from 7-ADCA and D-(-)-phenylglycine amide. The synthesis over hydrolysis ratios of the immobilized preparations were also much lower than that of the dissolved enzyme. This was partly due to diffusion limitations but also to an intrinsic property of the immobilized enzyme because the synthesis over hydrolysis ratio of the crushed preparations was much lower than that of the dissolved enzyme.

[Establishment of detective systems for GL-7ACA acylase expression].

Glutaryl-7-amino cephalosporanic acid (GL-7ACA) acylase catalyzes the conversion of GL-7ACA to 7-amino cephalosporanic acid (7-ACA). The product 7-ACA is a starting compound for semi-synthetic cephalosporin antibiotics in industry. In order to detect the expression and specific activity of protein-engineered GL-7ACA acylase accurately, two useful detective systems for its expression has been established, in which reporter genes xylE and lacZ were fused to the downstream the GL-7ACA acylase gene acy respectively and the activity of catechol dioxygenase or beta-galactosidase could indicate the amount of acy expression.

[Determination of degraded products of penicillin by high performance liquid chromatography].

A method for the determination of main composition in degraded products of penicillin, 6-aminopenicillanic acid (6-APA), penicillin G kalium (PGK), phenylacetic acid (PAA), and the by-product benzylpenicilloic acid (BPA) by HPLC was studied. Separation conditions were as follows: Spherisorb C18 column, 250 mm x 4.6 mm i.d., 10 micrograms; mobile phase: V (methanol): V(0.004 mol/L KH2PO4 buffer, pH 4.5) = 50:50; flow rate: 1 mL/min; detector: UV 230 nm. The method is effective, quick, accurate and reproducible. The satisfactory results show that this new method has certain practical values as an approach of quality control.

The use of chromogenic reference substrates for the kinetic analysis of penicillin acylases.

Determination of kinetic parameters of penicillin acylases for phenylacetylated compounds is complicated due to the low K(m) values for these substrates, the lack of a spectroscopic signal, and the strong product inhibition by phenylacetic acid. To overcome these difficulties, a spectrophotometric method was developed, with which kinetic parameters could be determined by measuring the effects on the hydrolysis of the chromogenic reference substrate 2-nitro-5-[(phenylacetyl)amino]benzoic acid (NIPAB). To that end, spectrophotometric progress curves with NIPAB in the absence and presence of the phenylacetylated substrates and their products were measured and analyzed by numerical fitting to the appropriate equations for competing substrates with product inhibition. This analysis yielded kinetic constants for phenylacetylated substrates such as penicillin G, which are in close agreement with those obtained in independent initial velocity experiments. Using NIPAB analogs with lower k(cat)/K(m) values, kinetic parameters for the hydrolysis of cephalexin and penicillin V were determined. This method was suitable for determining the kinetic constants of penicillin acylases in periplasmic extracts from Escherichia coli, Alcaligenes faecalis, and Kluyvera citrophila. The use of chromogenic reference substrates thus appears to be a rapid and reliable method for determining kinetic constants with various substrates and enzymes.

Specific substrates for spectrophotometric determination of penicillin acylase activity.

Penicillin acylase substrates suitable for colorimetric determination of the enzyme activity have been tested in this study. The kinetic parameters (Km and kcat) have been elucidated for the following nine substrates: six phenylacetic acid derivatives (p-nitroanilide, p-nitrophenyl ester, p-nitro-m-carboxyanilide, p-nitro-o-carboxyanilide, p-nitro-o-hydroxyanilide, m-nitro-p-carboxyanilide), two D-phenylglycine derivatives (p-nitroanilide, p-nitro-m-carboxyanilide), and also p-nitrophenyl ester of acetic acid (p-nitrophenyl acetate). With the exception of p-nitrophenyl acetate, all the compounds studied are highly specific chromogenic substrates for penicillin acylase, but their reactivity is very variable and kcat/Km values are in a range from 0.8.10(4) to 5.10(6) M(-1).sec(-1).

An efficient three steps preparative purification of penicillin acylase from Escherichia coli cells.

A new and efficient safe system for the purification of the penicillin acylase from Escherichia coli G271 is presented. It was found that after a selective precipitation with ammnonium sulphate, followed by two chromatographic steps (anion exchange followed by adsorption on hydroxyapatite support), the enzyme was enriched 98 times with a 100% activity recovery. An original way has also been used to study the chromatographic separation of the protein mixture in three major categories on DEAE resin, by an analysis of the concentrations of the different species in the breakthrough curve obtained from a complete saturation of the column.