Fig. 9. Analysis by SDS-PAGE of the selectivity of the immobilization of poly-His-tagged P-galactosidase on different Sepabeads supports. Lane 1, molecular weight markers; lane 2, crude preparation of P-galactosidase; lane 3, proteins that are not immobilized on IDA-Co-Sepabeads; lane 4, proteins that are desorbed from glycine-blocked IDA-Co-Sepabeads derivatives; lane 5, proteins that are not immobilized on standard Sepabeads; lane 6, proteins that are desorbed from glycine-blocked standard Sepabeads derivatives. The immobilized preparations were in all cases blocked with mercaptoethanol before the desorptions.
selective than the immobilization on the conventional one. Figure 9 shows that although many protein subunits could be detected using the standard support, only a band of 67 kDa (which corresponds to the molecular weight of the monomer of Htag-BgaA) could be detected in the bifunctional support.
3. Activity standard assay was performed with o-nitrophenyl- P-D-galactopyranoside (ONPG) at 25°C. ONPG was dissolved in Novo buffer, pH 6.5, and used at a final concentration of 13.3 mM
4. One unit of P-galactosidase activity is defined as the amount of enzyme that produces 1 |imol of o-nitrophenol per minute under the conditions described.
3.7. Structural Stabilization of Poly-His-Tagged P-Galactosidase From Thermus spp. Strain T2 on Sepabeads FP-EP IDA-Epoxy Via Multipoint Covalent Attachment
1. Immobilized enzymes derivative prepared on Sepabeads FP-EP IDA-epoxy were further incubated after covalent immobilization at pH 10.0 for 24 h. This was carried out to favored multipoint covalent attachment (see Subheading 3.5.). Figure 10 shows that this derivative of Htag-BgaA was more stable than the soluble enzyme and even more stable than that prepared using the conventional epoxy supports. Moreover, this immobilized derivative retained almost 100% of the initial activity after several weeks of incubation at 50°C (temperature employed normally at industrial pasteurization processes; see Fig. 11).
1. The epoxy supports (Sepabeads and Eupergit 250) must be stored dried at -20°C for the epoxy stability.
2. During covalent immobilization process, some enzymes may be inactivated at alkaline pH. In these cases, some inhibitors may be added to the immobilization/ incubation buffer to protect the enzyme.
3. Stirring should be perfomed gently to prevent support breakage. This may promote some fines where diffusion problems are decreased and activity may apparently increase.
4. The poly-His-tagged GA extract was produced in E. colias described previously (19).
5. Crude extract of poly-His-tagged P -galactosidase cloned and overexpressed in E. coli (MC1116) (Htag-BgaA) was produced as previously described (20). Only fresh enzyme preparations could be used to obtain immobilized enzyme derivatives with maximum loading. That results from the tendency of Htag-BgaA from Thermus strain T2 to self-associate in solution, and therefore to form large oligo-meric species that obstruct the pores of the matrix (21).
6. The Novo buffer is designed to reproduce the composition of milk (2.7 mMsodium citrate, 7.91 citric acid, 2.99 mM potassium biphosphate, 10.84 mM potassium phosphate, 19.43 mMpotassium hydroxide, 4.08 mMmagnesium chloride, 5.1 mM calcium chloride, and 3.33 mM sodium carbonate) (22).
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