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

The covalent immobilization of beta-galactosidase from Kluyveromyces lactis (beta-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 beta-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 lending 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 beta-gal). The K-app (3.4 mM) for the CDAP-derivative with ONPG as substrate was higher than the K-M value for soluble beta-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 beta-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 degrees 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. (C) 1998 Elsevier Science B.V. All rights reserved.