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.     

Effects of the loss of clonal integration on four sedges that differ in ramet aggregation

Although clonal growth is a dominant mode of plant growth in wetlands, the importance of clonal integration, resource sharing among ramets, to individual ramet generations (mother and daughter) and entire clones of coexisting species has not been well investigated. This study evaluated the significance of clonal integration in four sedge species of varying ramet aggregations, from clump-forming species (Clumpers –Carex sterilis, Eleocharis rostellata), with tightly aggregated ramets (rhizomes<1cm), to runner species (Runners –Schoenoplectus acutus, Cladium mariscoides), with loosely aggregated ramets. We manipulated clonal integration by either severing connections between target mother and daughter ramets or leaving connections intact, and then planted them in an intact neighborhood of a fen in Michigan, USA. We measured growth parameters of original and newly produced ramets over two growing seasons and conducted a final biomass harvest, to address four hypotheses. First, we expected integrated clones to accumulate more biomass than severed clones. However, final clone-level biomass and ramet production were the same for both treatments in all species although severing initially stimulated ramet production by Schoenoplectus and produced a more compact ramet aggregation in Cladium. Second, we hypothesized that mother ramets would experience a cost of integration, through reduced ramet or biomass production, while daughters would experience a benefit, through increased resource availability from mothers. Mother ramets of Cladium suffered a cost from integration, while Schoenoplectus mothers suffered a slight cost and Carex daughters saw a slight benefit. Finally, we hypothesized that integration would be more active in runner species than in clumper species. Indeed, we documented more active integration in runners than clumpers, but none of the study species were dependent upon integration for growth or survival once daughter ramets were established with their own roots and shoots. This study demonstrates that integration between established ramets may not be the most important advantage to clonal growth in this wetland field site. The loss of integration elicited varied responses among coexisting species in their natural habitat, somewhat but not completely related to their growth form, suggesting that a combination of plant life history traits contributes to the dependence upon clonal integration among established ramets of clonal species.     

Increased nucleophile reactivity of amino acid beta-naphthylamides in alpha-chymotrypsin-catalyzed peptide synthesis

Alpha-chymotrypsin-catalyzed acyl transfer from Boc-L-MetONp, Ac-L-TyrOEt, Bz-L-TyrOMe, Mal-L-PheOMe to the C-protected amino acids (L-AlaNH2, L-LeuNH2, L-ArgOMe and beta-naphthylamides of L-Arg, L-Leu, L-Ala and L-Glu) has been studied. Modification of the carboxylic groups with beta-naphthylamide was shown to increase the reactivity of nucleophiles in these reactions by a factor of more than 100 in comparison with amides and esters of the same amino acids. This effect can be accounted for by the effective formation of the nucleophile-acylenzyme complex due to hydrophobic interactions of the beta-naphthylamide moiety with the corresponding subsite of alpha-chymotrypsin. The reaction kinetics follows the scheme involving hydrolysis of the nucleophile-acylenzyme intermediate. The contribution of this pathway depends on the structures of both the acyl-group donor and the added nucleophile. The competitive inhibition by amino acid beta-naphthylamides is also observed. The results obtained show that modification of the COOH-group of added nucleophiles by beta-naphthylamide strongly affects the reactivity of these compounds in the alpha-chymotrypsin-catalyzed peptide synthesis.     

Determination of beta-mannanase activity by viscosimetric and spectrophotometric methods

The activity of beta-mannanase from Bacillus subtilis was measured viscosimetrically and spectrophotometrically. As substrate galactomannane of Ceratonia siliqua was used. Relationships between the beta-mannanase activity and the substrate concentration as well as the enzyme content were investigated. The kinetic parameters of the enzymes obeying the Michaelis-Menten equation were calculated. It was found viscosimetrically that Vmax of the commercial enzyme preparation was 1.4 mucat/g (at pH 5.8 and 40 degrees) and Km was 0.6 mM. The viscosimetric method shows high sensitivity, whereas the spectrophotometric technique suits mass-scale analyses.     

Lysine decarboxylase assay by the pH-stat method

A simple assay of lysine decarboxylase is described. It is based on measuring the rate of titration of OH- ions, released during decarboxylation with the aid of a pH-stat apparatus. The continuous recording of the reaction progress may be easily performed. Using the pH-stat method the Km for lysine decarboxylase from E. coli is 2.0 mM.     

Determination of initial velocities of enzymic reactions from progress curves.

The present communication describes a novel method for estimating initial velocities (v) of enzyme-catalysed reactions. It is based on an approximation of experimental data obtained by the cubic spline function. The initial velocity of a reaction is calculated as a derivative of the approximating function at a time value equal to zero. The proposed method is usable on a computer with a FORTRAN IV program. The method can be successfully used in such cases as substantial extents of substrate conversion, the inactivation of an enzyme in the course of a reaction, the existence of large experimental error or when the reaction mechanism is unknown.     

Choice of the initial enzyme correlation in immobilizing polyenzyme systems

An optimal, in respect to productivity (activity X stability), enzyme ratio for immobilization of multienzyme systems was calculated by using the kinetic parameters (KM and Vmax), data on the stability and yield of each enzyme during immobilization. The experimental data, obtained during combined immobilization of invertase, mutarotase and glucose oxidase, illustrate the theoretical propositions.     

Thermostability of glucose oxidase immobilized on silica gel by different methods

Immobilization and thermostability of glucose oxidase immobilized on silica gel MCA-750 7 by different methods were studied. The process of inactivation was found to follow two stages that differed in their rate. The most stable preparations were produced by immobilization based on bromacetyl and glutaraldehyde methods.