Petronella Catharina Raemakers-Franken

Metabolic Engineering toward Sustainable Production of Nylon-6

Nylon-6 is a bulk polymer used for many applications. It consists of the non-natural building block 6-aminocaproic acid, the linear form of caprolactam. Via a retro-synthetic approach, two synthetic pathways were identified for the fermentative production of 6-aminocaproic acid. Both pathways require yet unreported novel biocatalytic steps. We demonstrated proof of these bioconversions by in vitro enzyme assays with a set of selected candidate proteins expressed in Escherichia coli. One of the biosynthetic pathways starts with 2-oxoglutarate and contains bioconversions of the ketoacid elongation pathway known from methanogenic archaea. This pathway was selected for implementation in E. coli and yielded 6-aminocaproic acid at levels up to 160 mg/L in lab-scale batch fermentations. The total amount of 6-aminocaproic acid and related intermediates generated by this pathway exceeded 2 g/L in lab-scale fed-batch fermentations, indicating its potential for further optimization toward large-scale sustainable production of nylon-6.

Factors relevant to the production of (R)-(+)-glycidol (2,3-epoxy-1-propanol) from racemic glycidol by enantioselective oxidation with Acetobacter pasteurianus ATCC 12874

Acetobacter pasteurianus oxidizes glycidol with high activity, comparable to the oxidation of ethanol. The organism has a preference for the S-enantiomer, and the kinetic resolution process obeys a simple relationship, indicating an enantiomeric ratio (E) of 19. The compound is converted into glycidic acid, although a transient accumulation of glycidaldehyde occurs initially. Determination of other parameters revealed a temperature optimum of 50 degrees C, long-term stability (cells in the resting state), and a pH optimum compatible with the chemical stability of glycidol. However, it was also noted that respiration rates decrease at concentrations of glycidol above 1 M. This is most likely caused by substrate inhibition of the glycidol-oxidizing enzyme, the quinohemoprotein ethanol dehydrogenase. Comparison with existing methods for enantiomerically pure glycidol production indicated a number of attractive points for the method described here, although definitive evaluation must await further studies on the long-term stability under process conditions, reusability of the cells, and the mechanism of glycidol inhibition.

Clathrate-Type Complexation of Cephalosporins withβ-Naphthol

The cephalosporin antibiotics cephalexin, cephradine, cefaclor, and cefadroxil form clathrate-type complexes with β-naphthol in the solid state (depicted here). The crystal structures and packing forces are unraveled.

Chiral liquid chromatography–mass spectrometry for high-throughput screening of enzymatic racemase activity

In finding suitable biocatalysts for processes in chemical industry, expression libraries are constructed containing typically >10,000 clones. Search for a desired activity is done by examination of all the clones in one or more libraries using a high-throughput screening assay. Here we describe a method for the screening of the enzymatic racemase activity of clones from an expression library on alpha-amino-epsilon-caprolactam (ACL) using a fast chiral LC separation and ionspray-MS as the detection technique. After substrate incubation with S-ACL, the 96-well microplates were centrifuged to remove cell material. The conversion of S-ACL to R-ACL was monitored by quantitation of the R-ACL enantiomer. Separation of the two ACL enantiomers was performed on a Crownpak CR+ column within 1 min. A Gilson 215 autosampler with a 889 multiple injection probe was used for injecting the samples into the LC system. The total analysis time for a 96-well microplate was 56 min. The MS was operated in the positive-ion mode using selected ion monitoring at m/z 129 [M+H]+ of ACL. Using this method over 12,000 samples were analyzed without loss in performance of the system. The LC column remained stable without loss of resolution and the MS system did not show loss in sensitivity throughout the screening. Inter-day reproducibility was within 15%.

Cavities, Layers, and Channels in the Hosting Framework of Molecular Complexes Derived From Cephradine

The cephalosporin-type antibiotics Cephradine, Cephalexin, and Cefaclor form clathrate-type complexes with a variety of naphthalene derivatives. The crystal structures of these complexes are isomorphous. Interestingly, the hosting framework formed by these cephalosporins can adapt to the guest molecule. This phenomenon of induced-fit appears to have a much larger potential, with the consequence that a series of smaller compounds (such as benzene derivatives) as well as bulkier compounds can also be hosted by Cephradine. When benzene derivatives were used as guests, pronounced deviations in the antibiotic framework were observed, and it is possible to induce deviations strikingly different from those found for the complexes with the naphthalene derivatives. Evidently, the hosting structure formed by Cephradine is highly flexible. Hosting frameworks containing layers, channels, and various other types of cavities can be obtained by selection of an appropriate guest molecule. Remarkably, a number of structural features and interactions remain unaffected in all these antibiotic frameworks. These persistent features seem to delineate the boundaries of framework formation for these antibiotics, thus defining the scope of complex formation.

Flow injection analysis electrospray ionization mass spectrometry for high-throughput screening of a gene library for amidase activity

In high-throughput screening of gene and mutant libraries, high analysis speeds and short method development times are important factors. Mass spectrometry (MS) is considered to be a generic analytical technique with a relatively short development time. Furthermore, when applying flow injection analysis (FIA) for sample introduction, the requirements for high throughput are met. In this work, the use of a single quadrupole electrospray MS instrument for assaying amidase activity in a gene library is demonstrated. The desired selectivity for measuring the amino acid, the reaction product of the amidase reaction, in the presence of high concentrations of the corresponding amino acid amide substrate was obtained by selective ionization of the amino acid in negative ion mode electrospray. The only sample preparation required was a 200-fold dilution of the reaction mixture. For obtaining quantitative results, a complementary calibration procedure was set up to correct for the change in ionization suppression as a function of conversion. This approach was used to screen a Mycobacterium neoaurum gene library consisting of 11,520 clones with alpha-methylleucine amide as substrate within 24h. Conversion was measured on the [M-H]- species of the corresponding alpha-methylleucine (m/z 144). Five positive clones were detected with a conversion ranging from 0.2% to 3.4%.

Efficiency of cephalosporin complexation with aromatic compounds

The cephalosporin antibiotics cephradine, cephalexin, cefaclor and cefadroxil form complexes with β-naphthol and several other naphthalene derivatives. In these clathrate-type complexes, the cephalosporins form the host lattice for the naphthalene derivatives. Complexation with β-naphthol analogues can be employed to withdraw cephalosporins selectively from an aqueous solution. In this process, the most important parameter is the complexation efficiency, which expresses the extent to which the cephalosporins can be withdrawn from a solution. The complexation efficiencies for a series of guest molecules are explained in terms of both the thermodynamics of the complexation reaction and the structural features of the cephalosporin complexes. In this manner, insight is gained into the subtle relationship between the molecular structure of naphthalene derivatives and the stability of their complexes with the antibiotics. It is shown which molecular properties of the guest molecules are the most important ones for an optimal complexation efficiency of cephalosporins.

Induced fit phenomena in clathrate structures of cephalosporins

The antibiotics cephalexin, cephradine, cefaclor and cefadroxil form clathrate type inclusion compounds with naphthalene derivatives that readily crystallize from an aqueous solution. In these clathrates the antibiotic molecules form the hosting lattice and the naphthalene derivatives are the guest molecules, whereby water serves as “cement”. A list of potential guest molecules was drawn up using the concept of molecular similarity. This list was extended by a series of compounds which are not supposed to fit. It was shown that a large variety of naphthalene derivatives can be hosted in clathrates with cephalexin, cephradine and cefaclor. Cefadroxil, however, is much more selective in accommodating guest molecules. Although cephalexin, cephradine and cefaclor form the principal hosting lattice and govern the overall crystal structure of the clathrates, the guest molecules are capable of inducing deviations in the framework of the host molecules, i.e. induced fit. Cefadroxil, however, lacks this adaptability due to the rigid three-dimensional hydrogen bonded structure of its hosting framework, and an exact fit of a guest molecule in the hosting framework of cefadroxil is thus required, i.e. lock and key concept. All four antibiotics have a limited adaptability by varying the number of water molecules in the clathrates. Certain guest molecules replace water in order to obtain the required space for inclusion, whereas other guest molecules cause incorporation of extra water, which is apparently beneficial for the crystal packing. However, the adaptability due to varying the water content cannot account for the remarkable flexibility in accommodating guest molecules exhibited by cephalexin, cephradine and cefaclor. The concept of induced fit is relevant for the understanding and design of clathrate type structures.