Large-scale purification of enzymes

Abstract

Many standard procedures for the purification of proteins in the laboratory do not readily lend themselves to scaling up, whereas, on the other hand, some techniques relatively unsatisfactory in the laboratory are much more effective on a large scale. When producing gram or kilogram quantities of enzymes for use over an extended period, the storage properties and general tractability of the purified products become increasingly important. Hence enzymes from thermophilic sources frequently have advantages over those from mesophiles. The possible economic advantages of simultaneous large-scale multi-enzyme isolation over separate individual enzyme purifications are evaluated. Batchwise adsorption and elution from ion-exchange celluloses frequently replace traditional precipitation techniques in the early stages of a large-scale purification. Dialysis is replaced by concentration, dilution and reconcentration with the use of hollow-fibre ultrafiltration equipment. Antiphonally direct scaling-up of column chromatographic procedures is usually possible. Modifications to column geometry to maximize flow rates are often desirable but purification factors and recoveries comparable with those obtained on the laboratory scale can be achieved relatively easily. Classical affinity chromatographic techniques have not proved so amenable to large-scale work, mainly because of the enormous expense and rather short life of the matrices. However, the quasi-affinity chromatography afforded by the triazine dye conjugates has proved of great benefit. The materials are cheap to prepare. The coupling procedures are both simple and rapid and do not involve the use of noxious chemicals such as cyanogen bromide. Moreover the triazine linkage is more stable under a variety of conditions than the isourea formed in cyanogen bromide coupling. Considerable further exploitation of these versatile matrices is expected.