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Immobilization of β-galactosidase from Kluyveromyces lactis on silica and agarose: comparison of different methods
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...
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Published in: | Journal of molecular catalysis. B, Enzymatic Enzymatic, 1998-08, Vol.4 (5), p.313-327 |
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Main Authors: | , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | 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. |
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ISSN: | 1381-1177 1873-3158 |
DOI: | 10.1016/S1381-1177(98)00071-X |