Magdalena Klimek-Ochab

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.

Biocatalytic Resolution of Enantiomeric Mixtures of 1-Aminoethanephosphonic Acid

Several fungal strains, namely Bauveria bassiana, Cuninghamella echinulata, Aspergillus fumigatus, Penicillium crustosum and Cladosporium herbarum, were used as biocatalysts to resolve racemic mixtures of 1-aminoethanephosphonic acid using L/D amino acid oxidase activity. The course of reaction was analyzed by 31P-NMR in the presence of cyclodextrin used as chiral discriminating agent. The best result (42% e.e of R-isomer) was obtained with a strain of Cuninghamella echinulata.

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.

Application of the Beauveria bassiana strain for the enantioselective oxidation of the diethyl 1-hydroxy-1-phenylmethanephosphonate.

Biotransformation of diethyl 1-hydroxy-1-phenylmethanephosphonate using fungi Beauveria bassiana allowed resolving the racemic mixture of the substrate and due to the biocatalyst and reaction conditions modifications, leading to desired optical isomer.

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 cell-free extract proved to be distinct from bacterial pathway described before.

Lipases and whole cell biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid and its ester.

A wide spectrum of commercially available lipases and microbial whole cells catalysts were tested for biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1 and its butyryl ester. The best results were achieved for biocatalytic hydrolysis of ester: 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2 performed by lipase from Candida cylindracea, what gave optically active products with 85% enantiomeric excess, 50% conversion degree and enantioselectivity 32.9 for one pair of enantiomers. Also enzymatic systems of Penicillium minioluteum and Fusarium oxysporum were able to hydrolyze tested compound with high enantiomeric excess (68-93% ee), enantioselectivity (44 for one pair of enantiomers) and conversion degree about 50-55%. Enzymatic acylation of hydroxyphosphinate was successful in case when porcine pancreas lipase was used. After 4days of biotransformation the conversion reaches 45% but the enantiomeric enrichment of the isomers mixture do not exceed 43%. Obtained chiral compounds are valuable derivatizing agents for spectroscopic (NMR) evaluation of enantiomeric excess for particular compounds (e.g. amino acids).

A Simple and Green Procedure for the Microbial Effective Synthesis of 1-phenylethyl Alcohol in Both Enantiomeric Forms

Both R- and S-phenylethyl alcohol of high enantiomeric purity (98%) and with a satisfactory yield (40–80%) were obtained by bioreduction of acetophenone, catalyzed by whole cells of baker’s yeast.

Biodiversity in targeted metabolomics analysis of filamentous fungal pathogens by 1H NMR-based studies

The taxonomical classification among fungi kingdom in the last decades was evolved. In this work the targeted metabolomics study based on 1H NMR spectroscopy combined with chemometrics tools was reported to be useful for differentiation of three model of fungal strains, which represent various genus of Ascomycota (Aspergillus pallidofulvus, Fusarium oxysporum, Geotrichum candidum) were selected in order to perform metabolomics studies. Each tested species, revealed specific metabolic profile of primary endo-metabolites. The species of A. pallidofulvus is represented by the highest concentration of glycerol, glucitol and Unk5. While, F. oxysporum species is characterised by increased level of propylene glycol, ethanol, 4-aminobutyrate, succinate, xylose, Unk1 and Unk4. In G. candidum, 3-methyl-2-oxovalerate, glutamate, pyruvate, glutamine and citrate were elevated. Additionally, a detailed analysis of metabolic changes among A. pallidofulvus, F. oxysporum and G. candidum showed that A. pallidofulvus seems to be the most pathogenic fungi. The obtained results demonstrated that targeted metabolomics analysis could be utilized in the future as a supporting taxonomical tool for currently methods.Graphical Abstract

Direct determination of enantiomeric enrichment of chiral, underivatized aminophosphonic acids — useful for enantioselective bioconversion results evaluation

The possibility of applying 31P NMR spectroscopy for the determination of the enantiomeric excess of the racemic mixture of non-derivatized aminophosphonic acids with small side chains has been investigated. It is proven, that the effectiveness of the application of a chiral solvating agent strongly depends on the concentration of applied shift reagent and on the pH of the particular experiment. Effectual resolution protocols are elaborated for following phosphonic acids: 1-aminoethanephosphonic acid, 1-amino-2-methylpropanephosphonic acid, 1-aminophenylmethanephosphonic acid, 1-aminophenylmethane-phosphonic acid and 1-amino-2-phenylethanephosphonic acid.Graphical abstract

Fungal synthesis of chiral phosphonic synthetic platform – Scope and limitations of the method

Chiral hydroxyphosphonates due to their wide range of biological properties are industrially important chemicals. Chemical synthesis of their optical isomers is expensive, time consuming and not friendly to the environment, so biotransformations are under consideration. Among others, these compounds act as enzymes inhibitors. This makes the bioconversions of phosphonates, especially scaling experiments, hard to perform. Biocatalysis is one of the methods that can be applied in synthesis of optically pure compounds. To increase the efficiency of the process with whole cell biocatalysts, it is essential to ensure optimal reaction conditions that minimize cellular stress and can enhance the metabolic activity of cells. The present investigation focuses on the scaling up of the kinetic resolution of racemic mixture of 2-butyryloxy-2-(ethoxy-P-phenylphosphinyl)acetic acid, applying free and immobilized form of the fungal biocatalysts and two operation systems: shake flask and recirculated fixed-bed batch reactor. Protocols of effective mycelium immobilization on polyurethane foams were set for T. purpurogenus IAFB 2512, F. oxysporum, P. commune. The best results of biotransformation were obtained with the immobilized P. commune in the column recirculated fixed-bed batch reactor. The conversion reaches 56% (maximal for the kinetic process) and the enantiomeric enrichment of the isomers mixture ranges between 82 and 93% (93% for ester of RP,R conformation). All biocatalysts exhibit SP-preference toward tested compound, what is essential because of importance of the phosphorus atom chirality for its biological activity.