Development of a continuous solid phase process for reduction and thiol-dependent immobilization of yeast β-galactosidase

Reduction and covalent immobilization of Kluyveromyces lactis β-galactosidase through disulfide bonds onto thiolsulfinate-agarose was performed using two fixed-bed mini-reactors connected in series, one packed with thiopropyl-agarose (a solid phase reducing agent) and the other with thiolsulfinate-agarose (a thiol-reactive support). With the aim of optimizing the whole process, two reactor systems were assessed. In System I, the percolate from thiopropyl-agarose containing the reduced enzyme was re-circulated through the thiolsulfinate-agarose reactor alone. In System II, re-circulation was performed through both the reactors, improving the immobilization yield from 17% (System I) to 42% and the expressed activity from 25% (System I) to 56%. When the bio-reactor achieved with System II was fed with skimmed milk at 22 °C at a flow rate of 48 ml/h, steady state lactose hydrolysis reached 80%. In addition, it could be reused four times without losing its lactose hydrolysis capacity.