Preparation of enzymes from clarified solution

In many cases, especially when extracellular enzymes are being prepared for sale, the clarified solution is simply concentrated, preservative materials added, and sold as a solution or as a dried preparation. The concentration process chosen will be the cheapest which is compatible with the retention of enzyme activity. For some enzymes rotary evaporation can be considered, followed if necessary by spray drying. The most popular method, though, is ultrafiltration, whereby water and low molecular weight materials are removed by passage through a membrane under pressure, enzyme being retained. Ultrafiltration differs from conventional filtration and microfiltration with respect to the size of particles being retained (< 50 nm diameter). It uses asymmetric microporous membranes with a relatively dense but thin skin, containing pores, supported by a coarse strong substructure. Membranes possessing molecular weight cut-offs from 1000 to 100,000 and usable at pressure up to 2 MPa are available.

There are a number types of apparatus available. Stirred cells represent the simplest configuration of ultrafiltration cell. The membrane rests on a rigid support at the base of a cylindrical vessel which is equipped with a magnetic stirrer to combat concentration polarisation. It is not suitable for large scale use but is useful for preliminary studies and for the concentration of laboratory column eluates. Various large-scale units are available in which membranes are formed into wide diameter tubes (1 - 2 cm diameter) and the tubes grouped into cartridges. These are not as compact as capillary systems (area/volume about 25 m⁻¹) and are very expensive but are less liable to blockage by stray large particles in the feedstream. Cheaper thin-channel systems are available (area/volume about 500 m⁻¹) which use flat membrane sandwiches in filter press arrangements of various designs chosen to produce laminar flow across the membrane and minimise concentration polarisation. Capillary membranes represent a relatively cheap and increasingly popular type of ultrafiltration system which uses micro-tubular membranes 0.2 - 1.1 mm diameter and provides large membrane areas within a small unit volume (area/volume about 1000 m⁻¹). Membranes are usually mounted into modules for convenient manipulation. This configuration of membranes can be scaled up with ease. Commercial models are available that give ultrafiltration rates of up to 600 L hr⁻¹.

The steady improvement in the performance, durability and reliability of membranes has been a boon to enzyme technologists, encouraging wide use of the various ultrafiltration configurations. Problems with membrane blockage and fouling can usually be overcome by treatment of membranes with detergents, proteases or, with care, acids or alkalis. The initial cost of membranes remains considerable but modern membranes are durable and cost-effective. Ultrafiltration, done efficiently, results in little loss of enzyme activity. However, some configurations of apparatus, particularly in which solutions are recycled, can produce sufficient shear to damage some enzymes.