Directed evolution techniques have revolutionised strategies for enzyme development and improvement. Large libraries, containing typically > 1000 protein mutants, are pro duced and screened for desired properties (e.g. novel activity or improved stability). The correct application of selection pressure depends on the accuracy and robustness of the screen: hence direct screens are preferred to indirect screens. Protein stability is often screened indirectly by monitoring protein aggregation or resi dual activity after incubation at elevated temperatures. Both screens rely on irreversible inactivation of the protein upon unfolding. The assumption is made that positive 'hits' result from variants resistant to denaturation under the test conditions and do not take1 spontaneous protein refolding into account. We have established an affordable, high-throughput, direct screen for protein stability that can be automated. Building on previous work Edgell ct. al. (2003) Biochemistry. 42. 7587-7593 we have extended and optimised the technique for application entirely in the higher throughput niicroplate format. Pure target proteins are unfolded in niicrowells by serial addition of chemical denaturants such as urea or guanidine hydrochloride. The unfolding curves are recorded by measuring the changes in tryptophan fluorescence under equilibrium conditions. The midpoint of unfolding determined for each protein variant (CY2) is an indication of its stability. This screen was developed and tested using bovine and equine cytochrome r as well as preparations of fat-stabilised BSA. This screen requires high-throughput protein purification and suitable buffer conditions for stability analysis of each mutant. A multi-step process route, amenable to automation. has been developed that involves cell culturing. protein purification with metal affinity resin, buffer exchange by dialysis and unfolding analysis, all in a high-throughput format. factors taken into account are maximum cell density of micro-scale cell-culturing. target protein title, resin binding capacity and minimum final protein concentration required for stability screening. This route was developed using wild-type IIis-tagged transketola (E. coli tkt gene) over-expressed in E. coli IM107 pQR791. Transketolase mutants. D381A and Y440A. with decreased dimer stability were also designed as model systems for the stability analy sis. The reduced stability was clearly observed. The route needs more optimisation to yield sufficient transketolase required for accurate1 screening. Proper implementation of the process route will allow for screening of a thousand mutants per day for improved stability. The stabilities of proteins with 'hard to screen activities' and reversible folding can be improved more readily using this route.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

The microcellular polymer known as polyHIPE polymer (PHP), with modified physico-chemical characteristics, was developed as a cell matrix for the immobilization of the starch-degrading bacterium, Bacillus subtilis. Initially, a suspension of B. subtilis spores were inoculated in situ into a synthesized PHP matrix which had pore and interconnect sizes of 20 m and 5 m respectively. These inoculated spores were activated by supplying continuously well-aerated culture medium (LB medium) and placed in a 370C room for 24h incubation. On many occasions the PHP emulsions failed to polymerize in the presence of the spores and, when they did, the germination frequency of the spores was low. Since, the in situ entrapment of spores into synthesized PHP was unsuccessful, the immobilization technique was changed to the adsorption of spores onto the PHP matrix. This was performed by forced-flow inoculation of a suspension of pre-germinated spores onto PHP matrices sealed in a PTFE microchamber. Culture medium was pumped continuously through the matrix at a flow rate of 1.0ml/min for 24h and samples collected at various time intervals for the determination of the number of cells released from the PHP matrix and for a- amylase productivity. Two different of chemically structured of PHPs were used; vinyl-pyridine PHP (TVP) and sulphonated PHPs (SPHPs). The growth and enzyme productivity data were evaluated and compared. Three different pore and interconnect sizes of SPHPs were evaluated; 42.0±0.61mm, 36.0±0.50mm and 30.0±0.64 mm. The collected samples obtained from the 24h cultivation were used to determine α- amylase productivity and the loss of cells from the matrices. The data showed that sulphonated PHP had a better performance as a cell matrix compared to vinylpyridine PHP. The morphology, viability and proliferation of the immobilized cells on PHP matrices were observed by scanning electron microscopy (SEM). The SPHP with a pore size of 36.0mm performed better with respect to the production of α- amylase and the penetration of cells through the whole matrix compared to other SPHPs. Consequently, this matrix was selected and three different concentrations of LB medium (0.5x, 0.75x and 2x) and three different concentrations of cell loading (2 x 108/ml, 2 x 107/ml and 2 x 106/ml) were used to establish the - iii - relationships between yield, cell numbers, cell loading concentrations and media concentration. These results were compared with planktonic batch cell culture. The data showed that the total productivity of α-amylase enzyme produced by immobilized cells (on the basis of SPHP volume) was 7.6-fold higher than the planktonic batch culture. However, if productivity was determined on the basis of total volume of nutrient medium used, that of the immobilised cells was relatively low compared to planktonic batch culture. The effect of increasing the concentration of individual components of LB medium (tryptone /NaCl/ yeast extract) on the productivity of α-amylase and loss of cells from the matrices was also evaluated. Results showed that medium with double the yeast extract (D7) had the most significant impact on α-amylase production by B. subtilis cells for both systems; immobilized cells and planktonic batch cell culture. The overall yield of a-amylase by immobilized cell cultures was relatively low (<1) compared to planktonic batch cultures. However, the work from this thesis shows that the developed SPHP matrix could be used as a cell matrix but further studies are required to improve its productivity with respect to enzyme production.

No description available.