Intergeneric hybrid origin of the invasive tetraploid Cirsium vulgare.

The invasive tetraploid Cirsium vulgare hybridizes with both Cirsium and Lophiolepis. Its conflicted position in molecular phylogenies, and its peculiar combination of morphological, anatomical, and genomic features that are alternatively shared with representatives of Cirsium or Lophiolepis, strongly suggest its intergeneric hybrid origin. Genetic relationships of C. vulgare (8 samples) with genus Lophiolepis (11 species) and other representatives of genus Cirsium (12 species) were evaluated using restriction site-associated DNA sequencing (RADseq) and examined using analytical and imaging approaches, such as NeighborNet, Heatmap, and STRUCTURE, to identify nuclear genomes admixture. Estimation of the intensity of spontaneous hybridization within and between Cirsium and Lophiolepis was based on herbarium revisions and published data for all reported hybrids pertinent to taxa currently included in Cirsium or Lophiolepis. The genome of any examined Cirsium species is more similar to C. vulgare than to any Lophiolepis species, and vice versa. The nuclear genome of the tetraploid C. vulgare is composed of two equivalent parts, each attributable either to Lophiolepis or to Cirsium; the organellar RADseq data clustered C. vulgare with the genus Cirsium. Spontaneous hybridization between Cirsium and Lophiolepis is significantly less intensive than within these genera. Our analyses provide compelling evidence that the invasive species C. vulgare has an allotetraploid intergeneric origin, with the maternal parent from Cirsium and the paternal from Lophiolepis. For the purpose of delimiting monophyletic genera, we propose keeping Lophiolepis separate from Cirsium and segregating C. vulgare into the hybridogenous genus Ascalea.

Stability enhancement of Escherichia coli penicillin G acylase by glycosylation with yeast mannan.

Penicillin G acylase (PGA) from Escherichia coli was cross-linked with mannan dialdehydes. Conjugates were prepared with molecular masses varying from 140 to 580 kDa and containing from 18 to 50% (w/w) saccharides, the values depending on the reaction conditions (mannan/enzyme ratio), and by using mannans with different degrees of oxidation and weight-average molecular mass (M macro(w)). The pH- and thermo-stability of all preparations of glycosylated enzyme were improved remarkably, whereby the influence of the character of the linked mannan dialdehyde, its content, as well as the molecular mass of prepared glycoconjugates, on the stability of PGA, was evaluated. PGA glycosylated with the most oxidized mannan up to an M(w) of 490 kDa, containing 41% (w/w) saccharides, and retaining 90% of its original catalytic activity, showed the highest stability. The half-life of this PGA preparation increased significantly: 13-fold at pH 3, 7-fold at pH 10, and 3.5-fold at pH 8 (all at 37 degrees C), compared with the native enzyme. At higher temperatures (50 degrees C) even more significant stabilization was evident, a 16-fold increase in half-life, from 18 min to 289 min, at pH 8, being measured.

Stabilization of D-amino-acid oxidase from Trigonopsis variabilis by manganese dioxide.

Stabilization of immobilized D-amino-acid oxidase was achieved as follows. Yeast Trigonopsis variabilis producing D-amino-acid oxidase was used to deaminate cephalosporin C to glutaryl-7-aminocephalosporanic acid. Permeabilized cells were co-immobilized with manganese dioxide by entrapment in (poly)acrylamide gel so that hydrogen peroxide, liberated in the reaction, could be partially deactivated and both the enzyme and the substrate could be stabilized. Activity of entrapped cells was determined by HPLC and enzyme flow microcalorimetry. The process was evaluated in terms of activity, immobilization yield, storage stability and oxo-product formation by immobilized preparations. The storage stability of immobilized biocatalysts with MnO2 was nearly doubled and production of 2-oxoadipyl-7-aminocephalosporanic acid was 2-3-fold higher than by entrapped cells without MnO2. Glutaryl-7-aminocephalosporanic acid can be easily obtained from the resulting oxo-product by a non-enzymic reaction via externally added hydrogen peroxide.

Direct determination of the cephalosporin transforming activity of immobilized cells with use of an enzyme thermistor. 1. Verification of the mathematical model.

A simple method for the direct measurement of the catalytic properties of immobilized cells in the flow minicalorimeter, the enzyme thermistor (ET), is presented. A Trigonopsis variabilis strain with cephalosporin C-transforming activity was used as the model system. The yeast cells were immobilized either by crosslinking with a homobifunctional reagent or by entrapment in gels. The actual activity of the immobilized cells used in the ET was estimated by means of a stirred-batch reactor measurement in conjunction with HPLC analysis of substrate and products. Similar results were also obtained using D-amino acid oxidase (EC 1.4.3.3) isolated from T. variabilis cells and immobilized by gel entrapment. This calibration procedure was found to be appropriate for all biocatalyst systems used. The thermometric signal was proportional to the amount of biocatalyst immobilized in the ET minicolumn. It was shown that the rate of reaction catalyzed by T. variabilis entrapped in calcium pectate gel was limited by internal diffusion to an extent depending on the cell concentration in the biocatalyst particle. This approach offers a direct method for studying the kinetic properties of immobilized cells.