Determination of the Activity of Immobilized Biocatalysts in the Case of Poorly Soluble Substrates: The Example of D-5Phenylhydantoinase

The present communication describes the determination of activity of immobilized biocatalysts, from progress curves, in the case of a poorly soluble substrate. Computer simulation is used for the comparison of five well known methods for the determination of initial velocity of the reaction with the modified method of smoothing experimental data by cubic spline. The computer simulation data show that, in the case of poorly soluble substrate, it is expedient to use the method of data linearization in the co-ordinate system p/t versus p and method of splines.     

Coupled reactions for the determination of analytes and enzymes based on the use of luminescence

Immobilized enzymes are widely used in the clinical laboratory in the assay of several analytes and enzymes. The use of immobilized enzymes makes these reagents recoverable and re-usable, and in most cases increases their stability and catalytic activity. In conjunction with bioluminescent enzymes (firefly and bacterial luciferases) and chemiluminescent catalyst (peroxidase) we set up high-sensitive flow methods based on the use of nylon tube coil or epoxy methacrylate column as solid support. All the NAD(P)/NAD(P)H-dependent dehydrogenases (bacterial luciferase), ATP-dependent enzymes (firefly luciferase) and oxidases producing H₂O₂ (peroxidase) can be immobilized and a large variety of analytes have been sensitively measured. As an alternative format we also reported a dry chemistry method in which all the enzymes, substrates and cofactors are ready to use, supported on dry cellulose disks. Methodological problems such as flow conditions, stability, pH, ionic strength and analytical performances are also reported.     

Efficient chemoenzymatic synthesis of (S)-α-amino-4-fluorobenzeneacetic acid using immobilized penicillin amidase

An efficient chemoenzymatic route was developed for synthesis of (S)-α-amino-4-fluorobenzeneacetic acid, a valuable chiral intermediate of Aprepitant, using immobilized penicillin amidase catalyzed kinetic resolution of racemic N-phenylacetyl-4-fluorophenylglycine. The optimum temperature, pH and agitation rate of the reaction were determined to be 40 °C, 9.5 and 300 rpm, respectively. Kinetic resolution of 80 g L⁻¹ N-phenylacetyl-4-fluorophenylglycine by immobilized amidase 20 g L⁻¹ resulted in 49.9% conversion and >99.9% e.e. within 3 h. The unreacted N-phenylacetyl-4-fluorophenylglycine can be easily racemized and then recycled as substrate. The production of (S)-α-amino-4-fluorobenzeneacetic acid was further amplified in 1 L reaction system, affording excellent conversion (49.9%) and enantioselectivity (99.9%). This chemoenzymatic approach was demonstrated to be promising for industrial production of (S)-α-amino-4-fluorobenzeneacetic acid.     

Polymer supported synthesis of a natural product-inspired oxepane library

Natural product inspired compound collections are prevalidated due to the evolutionary selection of the natural product scaffolds. Their synthesis requires the development of novel strategies amenable to formats suitable for library build-up. We describe a method for the synthesis of an oxepane library inspired by the core structure of oxepane natural products endowed with multiple bioactivities. Core aspects of the strategy are the establishment of a one-pot method employing different immobilized scavengers, the employment of an enyne ring closing reaction and diversification by means of different transformations, for example, cycloadditions and cross-metathesis reactions. In total, a collection of 115 oxepanes was obtained in 5–6-step reaction sequences.     

Sequence-specific and DNA structure-dependent interactions of Escherichia coli MutS and human p53 with DNA

Many proteins involved in DNA repair systems interact with DNA that has structure altered from the typical B-form helix. Using magnetic beads to immobilize DNAs containing various types of structures, we evaluated the in vitro binding activities of two well-characterized DNA repair proteins, Escherichia coli MutS and human p53. E. coli MutS bound to double-stranded DNAs, with higher affinity for a G/T mismatch compared to a G/A mismatch and highest affinity for larger non-B-DNA structures. E. coli MutS bound best to DNA between pH 6 and 9. Experiments discriminated between modes of p53–DNA binding, and increasing ionic strength reduced p53 binding to nonspecific double-stranded DNA, but had minor effects on binding to consensus response sequences or single-stranded DNA. Compared to nonspecific DNA sequences, p53 bound with a higher affinity to mismatches and base insertions, while binding to various hairpin structures was similar to that observed to its consensus DNA sequence. For hairpins containing CTG repeats, the extent of p53 binding was proportional to the size of the repeat. In summary, using the flexibility of the magnetic bead separation assay we demonstrate that pH and ionic strength influence the binding of two DNA repair proteins to a variety of DNA structures.     

Effect of elevated atmospheric CO2 on mycorrhizal colonization, external mycorrhizal hyphal production and phosphorus inflow in Plantago lanceolata and Trifolium repens in association with the arbuscular mycorrhizal fungus Glomus mosseae

Plantago lanceolata and Trifolium repens were grown under ambient (400 μmol mol^–1) and elevated (650 μmol mol^–1) atmospheric CO₂ conditions. Plants were inoculated with the arbuscular mycorrhizal fungus Glomus mosseae and given a phosphorus supply in the form of bonemeal. Six sequential harvests were taken in order to determine whether the effect of elevated CO₂ on internal mycorrhizal colonization and external hyphal production was independent of the stimulatory effect of elevated CO₂ on plant growth. At a given time, elevated CO₂ increased the percentage of root length colonized (RLC), the total length of colonized root and the external mycorrhizal hyphal (EMH) density and decreased the ratio of EMH to total length of colonized root. When plant size was taken into account, the CO₂ effect on RLC and total length of colonized root was greatly reduced (and only apparent for early harvests in T. repens) and the effects on the EMH parameters disappeared. Root tissue P concentration was unchanged at elevated CO₂, but there was a decrease in shoot P at the later harvests. There was no direct effect of elevated CO₂ on P inflow for the earlier period (< 50 d) of the experiment. However, over the last period, there was a significant negative effect of elevated CO₂ on P inflow for both species, independent of plant size. It is concluded that elevated CO₂ had no direct effect on mycorrhizal colonization or external hyphal production, and that any observed effects on a time basis were due to faster growing plants at elevated CO₂. However, for older plants, elevated CO₂ had a direct negative effect on P inflow. This decrease in P inflow coincides with the observed decrease in shoot P concentration. This is discussed in terms of downregulation of photosynthesis often seen in elevated CO₂ grown plants, and the potential for mycorrhizas (via external hyphal turnover) to alleviate the phenomenon. The direction for future research is highlighted, especially in relation to carbon flow to and storage in the soil.     

Transmembrane electric potential difference of germ tubes of arbuscular mycorrhizal fungi responds to external stimuli

Measurements of the electric potential difference across the hyphal wall and the cell membrane were made on external hyphae of three species of arbuscular mycorrhizal fungus Gigaspora margarita , Scutellospora calospora and Glomus coronatum and on germ tubes of Gi. margarita . The values of transmembrane electric potential difference recorded (∼–40 mV) are less negative than those previously reported from hyphae of arbuscular mycorrhizal fungi closely associated with roots and from filamentous fungi. The external hyphae of arbuscular mycorrhizal fungi grown in soil had similar values of electric potential difference to those grown in soil-less culture, and to germ tubes. Thermodynamic calculations showed that despite these low values of electric potential difference, efficient high-affinity uptake of phosphate is possible. The transmembrane electric potential difference of germ tubes of Gi. margarita became more negative when plant root extract was added to the medium, showing for the first time that the early stages of interaction between plant and fungus occur via direct effects on the plasma membrane rather than via effects on gene expression. Addition of K⁺ reversibly depolarized the transmembrane electric potential difference of germ tubes of Gi. margarita , indicating that despite the low electric potential difference the fungus has control over the permeability of the plasmamembrane to K⁺.     

Identification and localization of calcium-dependent protease II inNeurospora crassa andUromyces appendiculatus

Summary The existence of Ca²⁺-dependent protease II in crude extracts ofNeurospora crassa andUromyces appendiculatus was demonstrated by immunoblotting using specific antibodies. In both extracts two immunoreacting bands were observed. The molecular mass of the major band inN. crassa corresponded to 37 kDa, while that inU. appendiculatus was 43 kDa, similar to that previously reported forAllomyces arbuscula. Immunofluorescence of the enzyme was predominantly localized in the apical regions of germlings and growing hyphae, suggesting a functional role for the enzyme in hyphal growth.