Epoxy Sepabeads: A Novel Epoxy Support for Stabilization of Industrial Enzymes via Very Intense Multipoint Covalent Attachment

Biotechnology Progress - Tập 18 Số 3 - Trang 629-634 - 2002
César Mateo1, Olga Abián2, Gloria Fernández‐Lorente2, Justo Pedroche2, Roberto Fernández‐Lafuente2, José M. Guisán2
1Departmento de Biocatálisis, Intituto de Catalisis, CSIC, Campus Universidad Autonoma, Cantoblanco, Italy.
2Departamento de Biocatalisis, Instituto de Catalisis, CSIC, Campus Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain

Tóm tắt

AbstractSepabeads‐EP (a new epoxy support) has been utilized to immobilize‐stabilize the enzyme penicillin G acylase (PGA) via multipoint covalent attachment. These supports are very robust and suitable for industrial purposes. Also, the internal geometry of the support is composed by cylindrical pores surrounded by the convex surfaces (this offers a good geometrical congruence for reaction with the enzyme), and it has a very high superficial density of epoxy groups (around 100 μmol/mL). These features should permit a very intense enzyme‐support interaction. However, the final stability of the immobilized enzyme is strictly dependent on the immobilization protocol. By using conventional immobilization protocols (neutral pH values, nonblockage of the support) the stability of the immobilized enzyme was quite similar to that achieved using Eupergit C to immobilize the PGA. However, when using a more sophisticated three‐step immobilization/stabilization/blockage procedure, the Sepabeads derivative was hundreds‐fold more stable than Eupergit C derivatives. The protocol used was as follows: (i) the enzyme was first covalently immobilized under very mild experimental conditions (e.g., pH 7.0 and 20 °C); (ii) the already immobilized enzyme was further incubated under more drastic conditions (higher pH values, long incubation periods, etc.) in order to “facilitate” the formation of new covalent linkages between the immobilized enzyme molecule and the support; (iii) the remaining epoxy groups of the support were blocked with very hydrophilic compounds to stop any additional interaction between the enzyme and the support. This third point was found to be critical for obtaining very stable enzymes: derivatives blocked with mercaptoethanol were much less stable than derivatives blocked with glycine or other amino acids. This was attributed to the better masking of the hydrophobicity of the support by the amino acids (having two charges).

Từ khóa


Tài liệu tham khảo

Bickerstaff G. F., 1997, Methods in Biotechnology 1

10.1016/0304-5102(86)85134-3

Gupta M. N., 1991, Thermostabilizationof proteins, Biotechnol. Appl. BioChem., 4, 1

10.1016/0167-7799(85)90104-0

10.1016/0167-7799(93)90080-S

Kennedy J. F., 1990, Immobilized enzymes and cells, Chem. Eng. Prog., 45, 81

10.1126/science.219.4585.722

Rosevear A., 1984, Immobilized biocatalysts‐A critical review, J. Chem. Technol. Bioctechnol., 34, 127, 10.1002/jctb.280340302

10.1080/03602458008066529

10.1016/S0141-0229(99)00188-X

10.1002/bit.10019

10.1016/S0021-9673(01)91648-6

10.1111/j.1470-8744.1988.tb00003.x

10.1016/0021-9673(93)80114-N

10.1016/S0021-9673(99)00484-7

Kramer D. M.;Lehmann K.;Pennewiss H.;Plainer H.Oxirane acrylic beads for protein immobilization: A novel matrix for biocatalysis and biospecific adsorption.26th International IUPAC Symposium on Macromolecules; 1979.

10.1016/1381-1169(95)00019-4

10.1016/S0141-0229(98)00028-3

10.1016/S0065-2164(08)70352-6

Gianfreda L., 1991, Enzyme stabilization: State of the art, Mol. Cell. BioChem., 109, 97

Nanalov R. J., 1993, Immobilization and properties of Bacillus stearothermophilus pulanase, Biotechnol. Appl. BioChem., 18, 409

10.1007/BF02788045

10.3109/07388559309041320

10.1016/0141-0229(88)90018-X

Guisan J. M., 1991, Immobilization‐stabilization of chymotrypsin by covalent attachment to aldehyde agarose gels, Biotechnol. Bioeng, 39, 75

10.1002/bit.260250804

10.3109/10242429508998152

10.1016/0141-0229(89)90019-7

Alvaro G., 1991, Immobilization‐stabilization of penicillin G acylase from E. Coli, Appl. Biochem. Biotechnol, 26, 210

10.1021/bm000071q

Thông tin
Thông tin xuất bản

Biotechnology Progress

Tập 18 Số 3

629-634

Thông tin tác giả