Bernd Niemeyer

Enzyme catalysis in organic solvents: influence of water content, solvent composition and temperature on Candida rugosa lipase catalyzed transesterification.

In the present study the influence of water content, solvent composition and reaction temperature on the transesterification of 1-phenylpropan-2-ol catalyzed by Candida rugosa lipase was examined. Reactions were carried out in different mixtures of hexane and tetrahydrofurane. The studies showed that an increasing water content of the organic solvent results in an increasing enzyme activity and a decreasing enantiomeric excess. Furthermore, a significant influence of the solvent hydrophilicity both on the enzyme activity and on the enantiomeric excess was found. An increase in solvent hydrophilicity leads to a decrease of enzyme activity and an increase of the enantiomeric excess. This indicates that the enzyme becomes more selective with decreasing flexibility. Similar effects were found by variation of the reaction temperature. Taken together, the decrease in conversion and the increase in selectivity with increasing solvent hydrophilicity are induced by the different water contents on the enzyme surface and not by the solvent itself.

Process development in affinity separation of glycoconjugates with lectins as ligands

Process development for affinity separation is a crucial prerequisite for a successful biospecific isolation of biological active substances like glycoconjugates or enzymes. The functionalization of polymer and silica based adsorbents and their influence on the adsorption behaviour of the modified adsorbents are presented. Improvement of the immobilization conditions for different lectins lead to a stable binding of more than 90% within 4 h of ligands applied to the immobilization solution. The prepared adsorbents are characterized according to specificity, stability and capacity. The isolation of the glycoprotein fetuin from fetal calf serum with wheat germ agglutinin adsorbents and the purification of horseradish peroxidase with concanavalin A (Con A) adsorbent are described. The Langmuir model, using glucose oxidase as glycoprotein and Con A adsorbents, expresses the sorption behaviour. The fixed bed separation is represented by the dispersion model. The process simulation supports the process development evaluating design parameters and investigating and optimizing process conditions. The influence of the flow as well as the concentration of contaminants competing with the valuable product for the ligands on the separation performance are demonstrated and discussed.

One‐step affinity purification of fetuin from fetal bovine serum

Fetuin is a plasma glycoprotein widely distributed in mammals. It has been used as a model protein for structural analyses and investigations into the biological properties of glycoproteins. A convenient one‐step procedure for biospecific isolation of fetuin from fetal bovine serum was developed on the basis of wheatgerm agglutinin (WGA) affinity separation. Two different porous supports, a silica‐based material and a polymer‐based material, were used for the immobilization of WGA. The prepared WGA adsorbents were characterized and process parameters of the affinity separation of fetuin were investigated and optimized. WGA was immobilized on silica and polymer supports with coupling yields of 99.6 and 99.4% respectively and amounts of coupled ligand of 7.9 and 9.2 mg of WGA/ml respectively. It has been shown that the specific capacities for fetuin were 5.1 mg/ml on WGA–silica, 1.8 mg/ml on WGA–polymer and 4.1 mg/ml on WGA–agarose. All three adsorbents proved to be suitable for the biospecific separation of fetuin. The polymer‐based WGA adsorbent was successfully applied in the purification of fetuin from fetal bovine serum in a one‐step separation process. The identity and purity of the isolated product was verified by SDS/PAGE. Under optimized conditions up to 21.6 mg of fetuin could be isolated from 1 ml of serum. The procedure described was designed to be easily scaled‐up for the production of fetuin.

Interaction of cellulose-based cationic polyelectrolytes with mucin.

Mucoadhesivity of water-soluble polymers is an important factor, when testing their suitability for controlled drug delivery systems. For this purpose, the interaction of new cationic cellulose polyelectrolytes with lyophilized mucin was investigated by means of turbidimetric titration, microscopy and measurement of zeta potential and particle size changes in the system. Results show that the cellulose derivatives interact with mucin. This interaction became stronger if cellulose macromolecules contained positively charged groups and an electrostatic interaction with the negatively charged mucin particles occurred. Under certain conditions flocculation of mucin particles by the cellulose polyelectrolyte was observed.

Preparation and characterization of PEGyated Concanavalin A for affinity chromatography with improved stability

In order to improve its stability, immobilized Concanavalin A (Con A) on Toyopearl adsorbents was conjugated with monomethoxy poly(ethylene glycol) succinimidyl propionate (mPEG-SPA) with different molecular weight. A colorimetric method using ninhydrin is proposed to determine the degree of PEGylation; this method has proved to be easy applicable and reproducible. The PEGylation reaction was studied in detail to elucidate how parameters such as molar ratio of mPEG-SPA to Con A and molecular weight of mPEG-SPA affect the degree of PEGylation. The adsorption isotherms of glucose oxidase (GOD) onto native and PEGylated Con A adsorbents showed that the modification did not alter substantially the specificity of the carbohydrate binding ability of Con A. However, the binding capacity for GOD was slightly reduced probably due to the steric hindrance caused by mPEG chains. Adsorption kinetic studies revealed a lower adsorption rate after PEGylation which was attributed to the steric effect. The dynamic adsorption capacity for modified Con A depended very much on the degree of PEGylation and the molecular weight of mPEG derivatives. The adsorption capacity could be highly preserved for Toyopearl Con A modified by mPEG2k (90% of the original adsorption capacity) even with a degree of PEGylation up to 20% (the ratio of primary amino groups of PEGylated immobilized Con A to that of native immobilized Con A). Studies show that the binding capacity of PEGylated Con A was highly preserved under mild process conditions. PEGylated Con A also exhibited obviously higher stability against more stressful conditions such as the exposure to organic solvents and high temperatures. Conjugation of Con A with mPEG2k provided better adsorption performance thus has greater potential for application in affinity separation processes compared with mPEG5k. The fact that PEGylation stabilizes the properties of Con A may greatly expand the range of applications of unstable proteins to bioprocessing (e.g. biocatalysis and downstream separation) as well as other protein applications (e.g. medication, industrial use, etc.).

Influence of reaction conditions on the enantioselectivity of biocatalyzed C–C bond formations under high pressure conditions

Benzoylformate decarboxylase (BFD, EC 4.1.1.7) is a homotetrameric thiamine diphosphate (ThDP)-dependent enzyme which catalyzes the synthesis of chiral 2-hydroxyketones accepting a broad range of aldehydes as substrates. In this study the synthesis of 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde was catalyzed by three BFD variants namely BFD F464I, BFD A460I and BFD A460I-F464I. This paper reports the effect of hydrostatic pressure up to 290 MPa when the reactions were carried out at different benzaldehyde concentrations (5-40 mM) as well as at different pH values (7.0-8.5). Acetaldehyde concentration was fixed at 400 mM in all biotransformations. Reactions performed at high benzaldehyde concentrations and at high hydrostatic pressures showed an increase in (R)-2-HPP formation catalyzed by all BFD variants. For BFD A460I-F464I we observed an increase in the ee of (R)-2-HPP up to 80%, whereas at atmospheric conditions this variant synthesizes (R)-2-HPP with an ee of only 50%. Alkaline conditions (up to pH 8.5) and high hydrostatic pressures resulted in an increase of (R)-2-HPP synthesis, especially in the case of BFD A460I and BFD F464I.

Influence of the Hydrostatic Pressure and pH on the Asymmetric 2-Hydroxyketone Formation Catalyzed by Pseudomonas putida Benzoylformate Decarboxylase and Variants Thereof

Benzoylformate decarboxylase (BFD) from Pseudomonas putida is a thiamine diphosphate‐dependent (ThDP) enzyme that catalyzes the asymmetric CC bond formation to (S)‐2‐hydroxypropiophenone [(S)‐HPP] starting from benzaldehyde and acetaldehyde. The enantioselectivity of BFD was shown to be a function of temperature and substrate concentration. It can additionally be changed by site‐directed mutagenesis on hot spot positions in the active site. In this article, we present the effect of hydrostatic pressure up to 250 MPa on the enantioselectivity for the recombinant wtBFD as well as for the variants BFD F464I, BFD A460I, and BFD A460I‐F464I. A general tendency toward lower amounts of (S)‐HPP could be observed at increasing pressures. For two of these variants an increase in pressure even caused an inversion in the enantioselectivity and thus increasing enantiomeric excesses, respectively. A pressure‐induced increase in enantioselectivity could therefore be observed for the first time in biocatalysis to the best of our knowledge. Furthermore, the pH is shown to be a parameter that also significantly influences the enantioselectivity of the reaction mentioned above. Biotechnol. Bioeng. 2010; 106: 18–26.

Test Gas Generation from Pure Liquids: An Application-Oriented Overview of Methods in a Nutshell

The generation of test gas from pure liquids has a wide variety of applications in laboratory and field experiments, for which the quality of the test gas is of significance. Therefore, various methods for test gas generation have been designed. Each method has unique advantages and disadvantages. Thus, a short overview is presented within the scope of this paper. Furthermore, a common bubbler system is presented, which was built to generate test gas from volatile organic compounds for experimental usage in laboratory applications. An analysis is conducted with respect to the generated concentrations at different temperatures and flow rates of the diluting gas. Accuracy and stability of this method are investigated.

CBRN Protection: Managing the Threat of Chemical, Biological, Radioactive and Nuclear Weapons

As an introductory text on NBC CBRN weapons and agents, this book leads the reader from the scientific basics to the current threats and strategies to prepare against them. After an introductory part on the history of NBC CBRN weapons and their international control, the three classes of nuclear/radiological, biological, and chemical weapons are introduced, focusing on agents and delivery vehicles. Current methods

Selective Adsorbents for Improved Removal of Malodorous Compounds

This work presents different methods of adsorbents characterisation. Their use towards improved removal of malodorous compounds is considered. These methods are applied to different modified silica gels. At first, the synthesis conditions were optimised using aminopropylsilanes. Afterwards the stability of the samples against ethanol and water was examined. The modified silica gels are suggested to be selective adsorbents in the field of odour control.