Expanded bed adsorption as a primary recovery step for the isolation of the insulin precursor MI3 process development and scale up.

Expanded bed adsorption (EBA) was evaluated for the isolation of the human insulin precursor MI3, expressed and secreted by the yeast Saccharomyces cerevisiae. The isoelectric point of the insulin precursor (pH 5.3) makes cation exchange a prime candidate for direct adsorption. In order to find a suitable window of operation for the process the adsorption equilibrium was analysed in a wide range of operating conditions (pH and conductivity) and for three different stationary phases. The same array of operating conditions was examined with regard to stable fluidisation of the adsorbents in S. cerevisiae suspensions. Interactions of the yeast with the fluidised stationary phase were investigated by a pulse response technique and the hydrodynamics of the fluidised bed under process conditions by residence time distribution analysis. The case study demonstrates that by parallel examination of product binding and fluidisation quality a window of operation can be found. Analysis of the binding kinetics by breakthrough experiments and modelling led to the definition of a set of operating conditions, which yield a compromise between optimal use of the equilibrium capacity provided by the adsorbent and high throughput required for an industrial separation. After initial experiments on the bench scale the protocol was transferred successfully to pilot scale demonstrating the design of a reliable operation.

(S,S)-2,3-Dihydroxy-2,3-dihydrobenzoic acid: microbial access with engineered cells of Escherichia coli and application as starting material in natural-product synthesis.

Cyclohexadiene-trans-5,6-diols such as (S,S)-2,3-dihydroxy-2,3-dihydrobenzoic acid (2,3-trans-CHD) have been shown to be of importance as chiral starting materials for the syntheses of bioactive substances, especially for the syntheses of carbasugars. By using methods of metabolic-pathway engineering, the Escherichia coli genes entB and entC, which encode isochorismatase and isochorismate synthase, were cloned and over-expressed in E. coli strains with a deficiency of entA, which encodes 2,3-dihydroxybenzoate synthase. A 30-fold increase in the corresponding EntB/EntC enzyme activities affects the accumulation of 2,3-trans-CHD in the cultivation medium. Although the strains did not contain deletions in chorismate-utilising pathways towards aromatic amino acids, neither chorismate nor any other metabolic intermediates were found as by-products. Fermentation of these strains in a 30 L pH-controlled stirred tank reactor showed that 2,3-trans-CHD could be obtained in concentrations of up to 4.6 g L(-1). This demonstrates that post-chorismate metabolites are accessible on a preparative scale by using techniques of metabolic-pathway engineering. Isolation and separation from fermentation salts could be performed economically in one step through anion-exchange chromatography or, alternatively, by reactive extraction. Starting from 2,3-trans-CHD as an example, we established short syntheses towards new carbasugar derivatives.