Arthur M. A. Pistorius

Monitoring of biomass composition from microbiological sources by means of FT‐IR spectroscopy

An FT‐IR spectroscopic method was developed for the simultaneous quantitative analysis of biomacromolecular components in biomass, originating from various microbiological sources. For the determination of protein, lipid and carbohydrate content, creatine phosphokinase, egg phosphatidyl choline and starch hydrolysate were chosen as external standards. This selection was based on spectral similarity and ease of availability. Protein content was based on the area under the amide II band profile around 1,545 cm−1. Because of the heterogeneous lipid composition in the different species, lipid content was determined using integration over the CH stretching vibrational population between 2,984 and 2,780 cm−1. Carbohydrate content was determined using integration over a CO and COC stretching band area between 1,180 and 1,133 cm−1. Linear regression analysis provided three calibration lines, according to which biomasses from ten species were analyzed. This approach showed good intra‐batch reproducibility. With this method we could demonstrate good reproducibility between batches of the same species with similar growth conditions while large differences in biomass composition were observed between the various species. Protein content as determined by FT‐IR spectroscopy compared well with the results obtained from elemental analysis. Biotechnol. Bioeng. 2009;103: 123–129.

pH dependence of copper geometry, reduction potential, and nitrite affinity in nitrite reductase

Many properties of copper-containing nitrite reductase are pH-dependent, such as gene expression, enzyme activity, and substrate affinity. Here we use x-ray diffraction to investigate the structural basis for the pH dependence of activity and nitrite affinity by examining the type 2 copper site and its immediate surroundings in nitrite reductase from Rhodobacter sphaeroides 2.4.3. At active pH the geometry of the substrate-free oxidized type 2 copper site shows a near perfect tetrahedral geometry as defined by the positions of its ligands. At higher pH values the most favorable copper site geometry is altered toward a more distorted tetrahedral geometry whereby the solvent ligand adopts a position opposite to that of the His-131 ligand. This pH-dependent variation in type 2 copper site geometry is discussed in light of recent computational results. When co-crystallized with substrate, nitrite is seen to bind in a bidentate fashion with its two oxygen atoms ligating the type 2 copper, overlapping with the positions occupied by the solvent ligand in the high and low pH structures. Fourier transformation infrared spectroscopy is used to assign the pH dependence of the binding of nitrite to the active site, and EPR spectroscopy is used to characterize the pH dependence of the reduction potential of the type 2 copper site. Taken together, these spectroscopic and structural observations help to explain the pH dependence of nitrite reductase, highlighting the subtle relationship between copper site geometry, nitrite affinity, and enzyme activity.

Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture.

Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling of complex recombinant proteins. Therefore, we evaluated several common yeast strains of the Saccharomycetoideae family as a source of high-quality, non-toxic yeastolates with the major aim to find a primary amino acid source for insect and mammalian cell culture that would allow cost-effective uniform stable isotope labelling (13C, 15N). Strains of the facultative methylotrophic yeasts Pichia pastoris and Hansenula polymorpha (Pichia angusta) as well as a strain of the baker’s yeast Saccharomyces cerevisiae were compared as a source of yeastolate with respect to processing, recovery and ability to sustain growth of insect and mammalian cell lines. The best growth-supporting yeastolates were prepared via autolysis from yeast obtained from fed-batch cultures that were terminated at the end of the logarithmic growth phase. Yeastolates obtained from H. polymorpha performed well as a component of insect cell cultures, while yeastolates from S. cerevisiae and H. polymorpha both yielded good results in mammalian cell cultures. Growth of yeasts in Heine’s medium without lactic acid allows relatively low concentrations of 13C and 15N sources, and this medium can be reused several times with supplementation of the 13C source only.

Tuning the supramolecular expression of chirality: phospholipid analogues containing amide linkages

The supramolecular expression of chirality in aggregates of amide-containing phospholipid analogues changes with the position and charge of the phosphate head group.

Assemblies of aziridinemethanols

Two novel long chain aziridinemethanols (1b, c) are described and their molecular organisation in the bulk and self-assembling properties in aqueous dispersion are reported. The orientation of the NH hydrogen of the aziridinealcohol moiety in 1b can be changed by introducing a methyl substituent into the rigid three-membered ring (1c), leading to a change in the hydrogen bonding pattern interconnecting these molecules. This change in configuration leads to marked differences in the ordering of these molecules in the solid state. Although compounds 1b and 1c both form highly organised structures in aqueous media and on the air–water interface, noteworthy differences are observed. Compound 1c yields left-handed helical ribbons whereas no chiral aggregates are found for 1b. However, the addition of 2-acetoxybenzoic acid (aspirin) to an aqueous dispersion of 1b leads to the generation of both left- and right-handed helical structures. Under these conditions a reaction had taken place that was specific for the ortho-isomer of acetoxybenzoic acid.

A single assay for multiple storage-sensitive red blood cell characteristics by means of infrared spectroscopy

BACKGROUND: To maintain a high quality of red blood cells (RBCs), RBC characteristics must be followed during storage under blood bank conditions. By means of infrared (IR) spectroscopy, several characteristics can be measured simultaneously.

IR, Biological Applications

Infrared spectroscopy can be applied for qualitative and quantitative analyses in all fields of biological research. This success is based on the high data acquisition speed, the possibility to use it noninvasively, and the absence of the need for staining or labeling. An infrared spectrum can be considered as a fingerprint, thus enabling its use for rapid identification of biological specimens by a spectral library search, cluster analysis, or artificial neural network analysis. Because of the complexity of biological samples, quantitative analysis nowadays is mostly carried out using a multiple linear regression approach such as partial least squares (PLS) regression. In these applications, the spectra are used as they are and focus is on the chemometric computational methods that are used after a spectrum is acquired. By deploying biochemical techniques such as site-directed mutagenesis and incorporation of stable isotope labels, one can introduce well-defined changes in an infrared spectrum, in aid of the interpretation of the observed spectral bands. Analysis of individual signals still can provide detailed information on a submolecular level.

Biological Applications of IR

Infrared spectroscopy can be applied for qualitative and quantitative analyses in all fields of biological research. This success is based on the high data acquisition speed, the possibility to use it noninvasively, and the absence of the need for staining or labeling. An infrared spectrum can be considered as a fingerprint, thus enabling its use for rapid identification of biological specimens by a spectral library search, cluster analysis, or artificial neural network analysis. Because of the complexity of biological samples, quantitative analysis nowadays is mostly carried out using a multiple linear regression approach such as partial least squares (PLS) regression. In these applications, the spectra are used as they are and focus is on the chemometric computational methods that are used after a spectrum is acquired. By deploying biochemical techniques such as site-directed mutagenesis and incorporation of stable isotope labels, one can introduce well-defined changes in an infrared spectrum, in aid of the interpretation of the observed spectral bands. Analysis of individual signals still can provide detailed information on a submolecular level.