assignment - Downstream processing Isolation and purification, the so called downstream processing, constitutes a key stage in the process

of production of biomolecules using microorganisms. This stage faces challenges from the fact that the target product forms only a minor part of a highly complex broth. Integration of downstream processing with fermentation The production of biomolecules during microbial fermentation is often limited by the phenomenon of product inhibition and also due to the undesirable activities such as proteases present in the culture broth. Integration of the product recovery step with that of fermentation has been a strategy employed for isolation of low molecular weight products which helps to improve the productivity as well as to reduce the number of unit operations required for purification. A model study using this approach has been done for lactic acid production by Lactobacillus casei cells. Integrating downstream processing for protein purification The major bottlenecks in the production of proteins are the elaborate and costly downstream processing required for getting the pure product and the unstable nature of the biomolecules. Addressing these issues comprise some of the goals of the research activities of the group. To limit the downstream processing costs, efforts are made to introduce separation techniques that are selective and that allow quick isolation of the biomolecules directly from the crude broth. This is achieved by integrating affinity interactions with a separation technique that can handle crude feeds and/or integrating the steps of broth clarfication, concentration and initial purification in one unit operation. The various separation techniques that are being studied are affinity precipitation, extraction in aqueous two-phase systems, displacement chromatography, shielded affinity chromatography, and expanded bed adsorption chromatography. Specificity in chromatography Affinity chromatography is of great interest since it allows the selective isolation of proteins. However, to use it for product isolation from a crude raw material, the choice of affinity ligands becomes limited. Robust, low molecular weight ligands are used. To improve the selectivity of separation using such a ligand, it is shielded by a complex with a weakly binding structure, thereby only allowing binding of molecules with high affinity. Competitive binding on the affinity support also facilitates elution of the captured material. Smart polymers as separation aids Stimuli responsive polymers or smart polymers are being extensively studied as means for facilitating separations. Stimuli such as small changes in pH, ionic strength or temperature, etc. alter the solubility of these polymers or polyelectrolyte complexes. The transition is completely reversible and precipitated polymer dissolves again on cooling or pH-change to its initial value. In affinity precipitation, the smart polymers are used as the carriers of affinity ligands. The soluble form of the polymer-ligand conjugate is used for interaction with the protein in the crude sample while precipitation is induced to effect separation. In aqueous two-phase extraction of proteins, use of smart polymers helps to solve one of the main problems of the technique, i.e. the separation of the protein from the phase-forming polymer, which is achieved simply by polymer precipitation by a moderate increase in temperature or pH-change. The easy separation of smart polymers from solution is exploited also when they are used as displacers in displacement chromatography. New procedures for elution from affinity chromatography are under development. When coupled to the chromatographic matrix, smart polymers change the properties of the matrix, e.g.

access of the protein molecules to affinity ligands, in response to small changes in temperature making it possible to control protein binding and elution by changing temperature without any changes in buffer composition. Since the area of smart polymers is rapidly expanding, synthesis and characterisation of new polymers is an important activity in the research group. Purification and characterization of enzymes from extremophiles A number of enzymes from alkaliphilic and cold-adapted organisms are purified for the purpose of characterization and comparison with corresponding enzymes from mesophiles. These include alkaline proteases, amylases, cyclodextrin glycosyltransferase, pectinase and -galactosidase. Purification is performed by both conventional and integrated separation techniques. Protein stabilization Proteins undergo denaturation during storage as a result of a variety of non-covalent and covalent reactions such as oxidation, deamidation, aggregation, etc., besides temperature. Use of stabilizing additives in protein formulations is the most common way of increasing the shelf life of the product. However, the choice of the additive is invariably made on an empirical basis. Our work so far has been based on the principle that a rational choice of the stabilizers can be made based on the knowledge of their protective effect against the specific denaturing reactions to which the protein is sensitive. We have, for example, shown that storage of an enzyme in the presence of both sorbitol and polyethyleneimine makes the moleceule resistant to inactivation by temperature and oxidation. Use is made of a number of biophysical techniques such as differential scanning calorimetry, circular dichroism, fluorescence spectroscopy and light scattering for facilitating these studies. Parallel to this, stability of enzymes from extremophilic microorganisms under conditions mimicking that of process environment is also determined. Structural adaptation with respect to stability under extreme conditions is of interest and so is the stability during application and storage.