Immobilization of β-galactosidase from Kluyveromyces lactis on silica and agarose: comparison of different methods

Abstract The covalent immobilization of β -galactosidase from Kluyveromyces lactis ( β -gal) on to two different porous carriers, CPC-silica and agarose, is reported. CPC-silica was silanizated and activated with glutaraldehyde. The activation of agarose via a cyanylating agent (CDAP) was optimized. Gel-bound protein and gel-bound activity were both measured directly, allowing the determination of apparent specific activities (S.A.). Higher amounts of β -gal were immobilized on the activated CPC-silica (maximum capacity, 23 mg ml −1 of packed support) than on the CDAP-activated agarose. For the lower enzyme loading assayed (12.6 mg ml −1 packed support), 100% of the enzyme was immobilized but only 34% of its activity was expressed. This inactivation during immobilization was confirmed by the S.A. values (22–29 EU mg −1 for the CPC-derivatives and 80 EU mg −1 for soluble β -gal). The K app (3.4 mM) for the CDAP-derivative with ONPG as substrate was higher than the K M value for soluble β -gal (2 mM). When the enzyme loading was increased five-fold, the K app increased four-fold, to 13 mM. The V app values for the CPC-derivatives were remarkably lower than the V max for soluble β -galactosidase. CDAP-derivatives showed better thermal stabilities than CPC-derivatives but neither of them enhanced the stability of the soluble enzyme. When stored at 4°C, the activity of both derivatives remained stable for at least 2 months. Both derivatives displayed high percentages of lactose conversion (90%) in packed bed mini-reactors. Glucose production was 3.3-fold higher for the CPC-derivative than for the CDAP-derivative, as a consequence of the higher flow rates achieved.