Metabolic production of a novel polymer feedstock, 3-carboxy muconate, from vanillin.

Vanillin can be produced on a commercial scale by depolymerising renewable lignin. One product of microbial metabolism of vanillin by common soil microbes, such as Acinetobacter baylyi, is a tricarboxylic acid with a butadiene backbone known as 3-carboxy muconate (3CM). Three enzymes, 4-hydroxy benzaldehyde dehydrogenase, vanillate monooxygenase and protocatechuate 3,4-dioxygenase, catalyse the biotransformation of vanillin to 3CM.

Tag removal by site-specific cleavage of recombinant fusion proteins.

Where an affinity tag has served its purpose it may become desirable to remove it from the protein of interest. This chapter describes the removal of such fusion partners from the intended protein product by cleavage with site-specific endoproteases. Methods to achieve proteolytic cleavage of the fusion proteins are provided, along with techniques for optimising the yield of authentic product.

The use of oxygen uptake rate to monitor discovery of microbial and enzymatic biocatalysts.

Arising from the requirement for discovery of novel biocatalysts with unusual properties, a process was developed which uniquely combines aspects of continuous culture with the measurement of oxygen uptake. This adaptation of the chemostat can be used to facilitate the isolation of a number of microorganisms with desirable properties, particularly those with useful metabolic capabilities and/or enzymes. The technique was also used to provide feedback on the metabolic status of a microbial population and increase the feed flow rate (i.

Immobilized metal ion affinity chromatography of histidine-tagged fusion proteins.

Immobilized metal ion affinity chromatography (IMAC) is a ubiquitous technique in modem recombinant production and purification. The wide range of expression vectors for the production of histidine-tagged recombinant proteins as well as the variety of stationary supports for their separation make IMAC an attractive and versatile choice for fast and reliable protein purification. It is not uncommon for IMAC purification to yield near homogenous target protein, with purities over 95%.

Immobilized metal ion affinity chromatography of native proteins.

Immobilized metal affinity chromatography (IMAC) is a common place technique in modem protein purification. IMAC is distinct from most other affinity chromatography technologies in that it can operate on a native, unmodified protein without the need for a specialized affinity "tag" to facilitate binding. This can be particularly important where a protein of interest is to be separated from a complex mixture such as serum or an environmental isolate.