Optimisation of isolation and purification of the jack bean enzyme urease by extraction and subsequent crystallization

A detailed investigation into the isolation by extraction and the purification by crystallization of the enzyme urease from jack bean meal is presented with a view to developing a large-scale process. The effect of different extraction solvents and additives upon the resulting crystals is characterised in terms of crystal size, protein yield and protein purity. The enzymatic activity of urease is exploited in order to quantify the amount and purity of the protein obtained. The extraction procedure was optimised and all stages of the process were monitored in order to provide as detailed a picture as possible of the impact of different process stages upon the intermediate products. Almost all protein is extracted in a single extraction step. Further extraction steps using the same meal but fresh solvent yield only small additional protein yields. Less than 10% of total protein is extracted in a second step, less than 3% in a third extraction. Different solvents lead to clear differences in product quality, with no single solvent optimising all quality criteria. In addition, the point at which the acetone precipitant is introduced into the process has a clear influence on the product. Judicious choice of extraction conditions can significantly increase the size of the urease crystals at the cost of product purity, quantified by specific activity. High product purity (a maximum 138-fold increase in purity was observed with a maximum yield of 8.4%), in contrast, leads to smaller crystals. Comparison to the purest commercially available urease revealed a similar urease content.