Comparative study of fungal cell disruption—scope and limitations of the methods

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems

A metal-independent hydrolase from a Penicillium oxalicum strain able to use phosphonoacetic acid as the only phosphorus source.

A Penicillium oxalicum strain was capable of the phosphate-sensitive utilization of phosphonoacetic acid as the sole source of phosphorus. A carbon-to-phosphorus bond-cleavage enzyme yielding acetic acid and inorganic phosphate was detected and characterized in extracts from cells grown on this phosphonate. Contrary to bacterial phosphonoacetate hydrolases, the fungal enzyme neither required nor was stimulated by divalent cations

Phosphonoacetate hydrolase from Penicillium oxalicum: purification and properties, phosphate starvation-independent expression, and partial sequencing.

The enzyme responsible for the hydrolysis of phosphonoacetic acid, a non-biogenic C-P compound, was purified to electrophoretic homogeneity from a wild-type strain of Penicillium oxalicum. A 50-fold enrichment was obtained by a combination of anion exchange, hydrophobic interaction and MonoQ-fast protein liquid chromatography, with a yield of one-third of the initial activity. A characterization of the protein showed both similarities and differences with respect to the well-characterized bacterial counterpart. The fungal phosphonoacetate hydrolase is a 43-kDa monomeric protein showing low affinity toward its substrate and high sensitivity to even mildly acidic pH values. Enzyme activity neither required nor was stimulated by the presence of divalent cations. Polyclonal antibodies were raised in mouse against the purified protein, allowing the study of enzyme induction as a function of the phosphate status of the cell. Peptide mass mapping led to the determination of about 20% of the primary structure. Despite the biochemical differences, amino acid alignment showed a high degree of similarity of the fungal hydrolase with the few sequences available to date for the bacterial enzyme. The possible physiological role of a phosphonoacetate hydrolase is discussed

Biocatalitic Transformation of epoxy- and vinylphosphonates

Przedmiotem badań była selektywna transformacja dwóch grup związków fosforoorganicznych epoksy- oraz winylofosfonianów do odpowiednich pochodnych, z wykorzystaniem całych komórek bakterii lub grzybów. Aktywność hydrolityczną względem modelowego epoksyfosfonianu wykazywał tylko szczep Aspergillus niger, podczas gdy szczepy Cladosporium herbarum oraz Fusarium oxysporum zdolne były do degradacji trwałego wiązania C-P w winylofosfonianach, w zadanych warunkach procesu.The subject of research was the selective transformation of the two groups of organophosphorus compounds, epoxy- and vinylphosphonates, to the corresponding derivatives, using whole cells of bacteria or fungi. Hydrolytic activity towards model epoxphosphonate was observed only in the case of Aspergillus niger, whereas Cladosporium herbarum and Fusarium oxysporum were able to degrade stable C-P bond in vinylphosphonates, under defined experimental conditions

Isolation and characterization of two new microbial strains capable of degradation of the naturally occurring organophosphonate–ciliatine

Air-born mixed fungal and bacterial culture capable of complete degradation of ciliatine was isolated. The utilization of the natural organophosphonate proceeded in the phosphate independent manner. Enzymatic activity involved in ciliatine degradation studied in the fungal cellfree extract proved to be distinct from bacterial pathway described before

Phosphonoacetic acid utilization by fungal isolates: occurrence and properties of a phosphonoacetate hydrolase in some penicillia

Among a collection of 18 fungal strains representing eight genera, only two strains (Penicillium oxalicum and P. minioluteum) were capable of growth on phosphonoacetic acid as sole phosphorous source. Enrichment liquid cultures in minimal medium with the compound as the only P-source selected four isolates, that were also identified as Penicillium spp. Phosphonoacetate metabolism did not lead to extracellular release of inorganic phosphate. In all cases phosphonoacetate hydrolase activity was detected in partially purified extracts, and a protein of the expected molecular mass reacted with polyclonal antibodies raised against the enzyme from P. oxalicum. There was no relation between phosphonoacetate hydrolase specific activity and growth rate or yield. Phosphonoacetic acid was the inducer of the hydrolase, independently of the concurrent availability of inorganic phosphate. Notwithstanding this, the utilization of the phosphonate was significantly inhibited in the presence of phosphate, suggesting an interference of the latter with phosphonoacetic acid uptake