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

BETA-GALATOSIDASA

INMOBILIZACION DE ENZIMAS

KLUYVEROMYCES LACTIS

HIDROLISIS

LACTOSA

BIBLIOGRAFIA NACIONAL QUIMICA

1998

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 Kapp (3.4 mM) for the CDAP-derivative with ONPG as substrate was higher than the KM value for soluble β-gal (2 mM). When the enzyme loading was increased five-fold, the Kapp increased four-fold, to 13 mM. The Vapp values for the CPC-derivatives were remarkably lower than the Vmax 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.

Journal of Molecular Catalysis B:Enzymatic v. 4, 1998. -- p. 313-327

Elsevier

1998

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doi:10.1016/S1381-1177(98)00071-X

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