Design and optimization of membrane chromatography process for monoclonal antibody charge variant separation

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Integrated supercritical fluid extraction and bioprocessing

ABSTRACT Supercritical fluids are increasingly being used and promoted at a laboratory and pilot scale to produce high value, natural bioactives from biologically based raw materials. Supercritical CO 2 is overwhelmingly the solvent of choice for these operations, but is largely limited to the processing of dry raw materials and the extraction of low polarity, low molecular weight compounds. The use of co-solvents and the use of alternative 'near-critical' extraction fluids such as dimethyl ether show potential to mitigate these limitations. Commercialisation of new supercritical extraction processes has arguably been limited because the supercritical extraction process has been developed in isolation of other processing steps necessary to achieve a successful product. This study reviews recent developments in integrated processing that incorporate the use of supercritical fluids for bioseparations and in particular process schemes that produce high value natural bioactives. Integrated processes include prior operation (fermentation, extraction, enzyme pre-treatment, physical fractionation or size reduction) followed by supercritical extraction or fractionation and processes in which operations are carried out in situ in supercritical fluids (supercritical chromatography, enzymatic conversion, precipitation and coating of solutes). The use of co-solvents and alternative extraction solvents in these processes is discussed. Prospects for future developments are also discussed

Lipase mediated simultaneous esterification and epoxidation of oleic acid for the production of alkylepoxystearates

Epoxy alkylstearates were synthesized by lipase catalysed esterification and perhydrolysis followed by epoxidation of oleic acid in a one-pot process. Immobilized Candida antarctica lipase (Novozym 435) was used as the catalyst. The esterification reaction occurred relatively quickly and was followed by epoxidation of the alkyl ester and the remaining fatty acid. Higher degree of esterification was achieved with n-octanol, n-hexanol and n-butanol as compared to that with ethanol and iso-propanol. The rate and yield of epoxidation was enhanced with iso-propanol but was lowered with the other alcohols. The lipase suffered significant loss in activity during the reaction primarily due to hydrogen peroxide. The presence of alcohols, in particular ethanol. further contributed to the enzyme inactivation. The epoxidation reaction could be improved by step-wise addition of the lipase. (c) 2006 Elsevier B.V All rights reserved

Characterisation of the First Enzymes Committed to Lysine Biosynthesis in <em>Arabidopsis thaliana</em>

<div><p>In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from <em>Arabidopsis thaliana</em>. The <em>A. thaliana</em> DHDPS enzyme (<em>At</em>-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (<em>S</em>)-lysine. Structural studies of <em>At</em>-DHDPS2 show (<em>S</em>)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from <em>A. thaliana</em> (<em>At</em>-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that <em>At</em>-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but <em>ab initio</em> modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.</p> </div

Analysis of DHDPR interface regions using the PDBePISA web server (<i>38</i>).

<p>Analysis of DHDPR interface regions using the PDBePISA web server (<i>38</i>).</p

Crystal structures of unliganded and lysine bound <i>At</i>-DHDPS2.

<p>A) Wall-eyed stereo image of the Cα superposition of <i>At-</i>DHDPS2 with bound lysine (blue Cα trace) and unliganded <i>At-</i>DHDPS2 (gold Cα trace; rmsd = 0.3 Å). The lysine molecules bound at the allosteric site of each monomer of the tetramer are shown in yellow (stick representation). B) The lysine binding site at the monomer-monomer interface of the tight-dimer showing residues in contact with the bound lysine molecules (yellow). Electron density around the bound lysine (grey mesh, contoured at 1.0 sigma) was calculated using refined coordinates omitting the bound lysine molecules. Residues contributed by each monomer of the tight-dimer are shown in different shades of blue, and are indicated by the use of the prime (’) symbol. C) overlay of the lysine binding residues of the tight-dimer from the lysine bound (blue) and unliganded (gold) structures. Lysine molecules are shown in yellow. Residues contributed by each monomer of the tight-dimer are shown in different shades of blue or gold, and are indicated by the use of the prime (’) symbol.</p

X-Ray scattering data of <i>At</i>-DHDPS2.

<p>Data were collected in the absence of ligand, or in the presence of 1 mM (S)-lysine, top panel; curves have been arbitrarily displaced along the logarithmic axis for clarity. Solid lines show the scattering profile from the unliganded crystal structure, calculated using CRYSOL. Distance-distribution functions, <i>p(r)</i> for the unbound and ligand bound <i>At</i>-DHDPS2 were determined using the indirect Fourier tranformation package GNOM (bottom panel).</p

X-Ray scattering of DHDPR.

<p>Data were collected for <i>At</i>-DHDPR2, <i>Ec</i>-DHDPR and <i>Tm</i>-DHDPR (panel A); curves have been arbitrarily displaced along the logarithmic axis for clarity. Data was analysed using GNOM (fitted data shown by red line in panel A) to calculate a distance distribution function for each enzyme (panel B).</p