Michael K. Turner

A study of the effect of specific growth rate and acetate on recombinant protein production of Escherichia coli JM107

The growth of Escherichia coli in fed-batch and continuous culture is examined and results show that α-amylase production is strongly dependent on specific growth rate (dilution rate) of the culture and production is greatest at an intermediate rate. Using continuous culture, it has also been found that the presence of acetate above a certain concentration reduces both μmax, and the production of recombinant protein.

Candida cloacae oxidation of long chain fatty acids to dioic acids

Candida cloacae cells oxidize long-chain fatty acids to their corresponding dicarboxylic acids (dioic acids) at rates dependent on their chain length and degree of saturation. This is despite the well-known toxicity of the fatty acids. Among the saturated substrates, the oxidation is limited to lauric acid (C12). The addition of pristane (5% v/v), which acts as an inert carrier for the poorly water-soluble substrate, boosts the oxidation of lauric acid to a rate that is comparable to that of dodecane. When dissolved in pristane, myristic (C14) and palmitic (C16) acids are effective carbon sources for C. cloacae, but dioic acid production is very low. Media glucose concentration and pH also influence cell growth and productivity. After the glucose is depleted, oxidation is optimal at a low pH. A two-phase (pristane/water) reaction was tested in a 2-l stirred tank bioreactor in which growth and oxidation were separated. A 50% w/w conversion of lauric acid (10 g/l) to dodecanedioic acid was achieved. The bioreactor also alleviated poor mass transfer characteristics experienced in shake flasks.

Principal component analysis of nonlinear chromatography.

Principal component analysis (PCA) has been used for the modeling of nonlinear chromatography under overload conditions. A 10‐fold range of crude erythromycin samples were loaded onto columns with different stationary‐phase chemistries (2 polystyrene, 1 methacrylate) in direct proportion to the bed volumes. The elution profiles indicated slightly concave isotherms for the polystyrene supports and a convex Langmuirian isotherm for the methacrylic support used. The principal component models accounted for over 98% of the original variance in the data for all three columns and were able to give excellent models of complete chromatograms in the absence of first‐principle models or physicochemical data. Correlations between sample mass and the principal component scores were made for each that were consistent for the column types despite the different geometries and stationary phases. Linear relationships with high correlation coefficients were observed when the scores of the same principal component were compared between columns. Such correlations offer considerable potential for modeling of nonlinear chromatography.

Graphical method for the calculation of chromatographic performance in representing the trade-off between purity and recovery

A simple engineering framework that enables the rapid representation of the performance of liquid chromatographic separations is provided in this paper. The fractionation diagram and its associated maximum purification factor versus product yield, and contamination index versus product yield diagrams, may be derived directly from chromatographic data. The fractionation diagram plots the relative change in the cumulative fractional mass of product eluted with the corresponding fractional total mass eluted, while the maximum purification factor versus yield diagram shows the degree of trade-off between the levels of purity and recovery achieved in the chromatographic step. The minimum contamination index versus yield plot is especially suitable for cases where the product and impurity are expressed in different units and shows how the extent of contaminant removal changes relative to product yield. These diagrams are more straightforward and easily interpretable compared to the basic conventional chromatograms and enable investigation of the degree of trade-off between purity and recovery for any set of operating conditions to be made. The approach is demonstrated for two different chromatographic systems. In the first, a set of simulation results from a verified size exclusion model is used to demonstrate the approach for product recovery. In the second, a set of experimental results for the removal of endotoxin from DNA is used. This demonstrates a problem where the product and impurity content are measured by different assay techniques and are expressed in different units, and also where the quality of process information is limited by the small number of fractions collected. The studies show how such an approach can help to identify the optimal operating conditions, in terms of acceptable yield and desired level of contaminant removal, and to redefine the location of product fractions needed to achieve these specifications.

Mechanical Stability of Immobilized Biocatalysts (CLECs) in Dilute Agitated Suspensions

Cross‐linked enzyme crystals (CLECs) are a novel form of immobilized biocatalyst designed for application in industrial biotransformation processes. In this work we have investigated the mechanical stability of agitated CLEC suspensions in relation to the design and scale‐up of bioconversions carried out in stirred‐tank reactors. By careful control of the crystallization conditions yeast alcohol dehydrogenase I (YADHI) microcrystals of different size were first prepared having either an hexagonal (∼12 μm) or rod‐shaped (∼4.6 μm) morphology. These were then cross‐linked with glutaraldehyde to form CLECs. The rate of breakage of the CLEC suspensions was subsequently measured in a rotating disk shear device (total volume, 11 mL) by monitoring the change in crystal size distribution with time. This device is designed to mimic the shear and energy dissipation rates found in a range of process scale equipment and may be used to study the mechanical stability of any immobilized biocatalyst preparation. Experiments were performed as a function of the speed and duration of disk rotation, CLEC concentration (0.26–2.5 mg·mL−1) and energy dissipation rate (2.2 × 103 to 6.8 × 105 W·kg−1). No breakage of the rod‐shaped CLECs was observed over the entire range of experimental conditions investigated. Breakage of the larger hexagonal‐shaped CLECs did occur, however, at energy dissipation rates, ϵmax, above 1.0 × 105 W·kg−1, where the calculated length scale of turbulence was around 2.0 μm. Based on visual observation of the sheared CLEC suspensions and models of crystal breakage, it was concluded that breakage of the hexagonal‐shaped CLECs occurred due to shear induced attrition. Measurement of the catalytic activity of both the hexagonal and rod‐shaped CLECs showed no significant change in activity before and after shearing.

Biocatalysis in organic chemistry (Part II): present and future

Biocatalysis has an established role in the manufacture of organic chemicals: glucose isomerase catalyses the manufacture of about 8 × 10 6 tonnes of isoglucose each year, and the annual output of many other reactions exceeds 10 3 tonnes. However, there is a need for a more-varied range of catalysts, and future developments in polymer synthesis may lead to the creation of enzymes that display broader specificity and selectivity. Whether catalysts are isolated from natural sources or are produced chemically, the engineering required to create an efficient and economic process is crucial. New developments should allow the technology to extend well beyond its present base in the pharmaceutical industry.

Biocatalysis in organic chemistry (Part I): past and present

Methods used in the manufacture of both ethanol and ascorbic acid have been developed over the past 150 years. The early stages of the development of both processes were characterized by the interaction between chemists, biologists and engineers, but social and economic influences also played their part. The history of these two examples illustrates the diversity of skills and influences needed for the application of biological catalysis to large-scale chemical manufacture.

Pharmaceuticals from agriculture: manufacture of discovery?

Abstract Agriculture competes with microbiology and experimental biochemistry as a resource for the discovery and synthesis of new drugs. In contrast, little of the benefit of legal manufacture returns to agriculture. Plants may lay claim to the input raw material in 25% of all prescriptions, but the safety, added value and efficacy of the drugs themselves are created by the pharmaceutical and fine chemical industries. These industries often replace the agricultural input with material from petrochemical sources, and those which remain are either small in volume or are byproducts of a large volume process. High product costs reflect difficult purifications and extensive chemistry rather than a genuine agricultural value. Modern developments with transgenic animals and plants are unlikely to change this situation. Even where the pharmaceutical industry finds plants or large animals useful for the manufacture of some products, the production itself is likely to stay within the pharmaceutical industry, if only to ensure adequate standards of safety and hygiene. Moreover, the same techniques which allow the biosynthesis of some products to be transferred to plants and animals allow the transfer of others back to cells suitable for growth in fermenters, allowing the fine chemical industry to replace even more of the agricultural output which enters the pharmaceutical industry.