The response effect of pheochromocytoma (PC12) cell lines to oxidized multi-walled carbon nanotubes (o-MWCMTs).

BACKGROUND: The applications of oxidized carbon nanotubes (o-CNTs) have shown potentials in novel drug delivery including the brain which is usually a challenge. This underscores the importance to study its potential toxic effect in animals. Despite being a promising tool for biomedical applications little is known about the safety of drugs in treating brain diseases. The toxicity of oxidized multi-walled carbon nanotubes (o-MWCNTs) are of utmost concern and in most in-vitro studies conducted so far are on dendritic cell (DC) lines with limited data on PC12 cell lines. OBJECTIVES: We focused on the effect of o-MWCNTs in PC12 cells in vitro: a common model cell for neurotoxicity. METHODS: The pristine multi-walled carbon nanotubes (p-MWCNTs) were produced by the swirled floating catalytic chemical vapour deposition method (SFCCVD). The p-MWCNTs were then oxidized using purified H2SO4/HNO3 (3:1v/v) and 30% HNO3 acids to produce o-MWCNTs. The Brunauer-Emmett-Teller (BET), transmission electron microscopy (TEM), Scanning electron microscopy (SEM), thermogravimetric analyser (TGA) and Raman spectroscopy techniques were used to characterize the MWCNTs. The PC12 cells were cultured in RPMI medium containing concentrations of o-MWCNTs ranging from 50 to 200 µg/ml. RESULTS: The o-MWCNTs demonstrated slight cytotoxicity at short time period to PC12 neuronal cells whilst at longer time period, no significant (p > 0.05) toxicity was observed due to cell recovery. CONCLUSION: In conclusion, the o-MWCNTs did not affect the growth rate and viability of the PC12 cells due to lack of considerable toxicity in the cells during the observed time period but further investigations are required to determine cell recovery mechanism.

A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening.

Hydroxynitrile lyases (Hnls) are important biocatalysts for the synthesis of optically pure cyanohydrins, which are used as precursors and building blocks for a wide range of high price fine chemicals. Although two Hnl enzymes, from the tropical rubber tree Hevea brasiliensis and from the almond tree Prunus amygdalus, are already used for large scale industrial applications, the enzymes still need to be improved and adapted to the special demands of industrial processes. In many cases directed evolution has been the method of choice to improve enzymes, which are applied as industrial biocatalysts. The screening procedure is the most crucial point in every directed evolution experiment. Herein, we describe the successful development of a novel screening assay for Hnls and its application in high-throughput screening of Escherichia coli mutant libraries. The new assay allows rapid screening of mutant libraries and facilitates the discovery of improved enzyme variants. Hnls catalyze the cleavage of cyanohydrins to hydrocyanic acid and the corresponding aldehyde or ketone. The enzyme assay is based on the detection of hydrocyanic acid produced, making it an all-purpose screening assay, without restriction to any kind of substrate. The gaseous HCN liberated within the Hnl reaction is detected by a visible colorimetric reaction. The facile, highly sensitive and reproducible screening method was validated by identifying new enzyme variants with novel substrate specificities.

High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts.

(S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis catalyzes the formation of (S)-cyanohydrins from hydrocyanic acid and aldehydes or ketones. This enzyme accepts aliphatic, aromatic, and heterocyclic carbonyl compounds as substrates and is therefore considered a potent biocatalyst for the industrial production of optically active chemicals. Limitations in enzyme supply from natural resources were overcome by production of the enzyme in the microbial host systems Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. Expression of Hnl in the prokaryotic system led to the formation of inclusion bodies whereas in both yeast hosts high levels of soluble protein were obtained. Highest yields were obtained in a high cell density batch fermentation of a P. pastoris transformant that expressed heterologous Hnl to about 50% of the soluble cytosolic protein. At a cell density of 100 g/liter cell dry weight, a volume yield of 22 g/liter of heterologous product was obtained. Attempts to produce the Hnl protein extracellularly with the yeast hosts by applying different leader peptide strategies were not successful. Immunofluorescence microscopy studies indicated that the secretion-directed heterologous Hnl protein accumulated in the plasma membrane forming aggregated clusters of inactive protein.