Notes

1. According to Axen et al. (9) it is possible to regulate the degree of thiol agarose substitution (thiol content) by varying the amount of epichlorohydrin used in the epoxy activation step.

2. This reaction is highly dependent on temperature and on incubation time. It is possible to decrease incubation time by working at 60°C.

3. It is possible to confirm the highly charged properties of the Bunte-salt gel by observing the increase in the swelling and in the volume of the gel.

4. Reduction of the Bunte-salt gel is carried out by using a small excess of DTT (e.g., 10%) with respect to the stoichiometric amount. This is important because of the high cost of this reagent.

5. A higher content of thiol groups (e.g., 1000 pmoles per gram of dried gel) would be desirable if the thiol-agarose is intended for use as a solid-phase reducing agent.

6. Longer incubation periods lead to a loss of activated groups (6).

7. Magnesium monoperoxyphthalate hexahydrate (MMPP) is a cheap and safe oxidant which can be purchased from Merck Schuchardt (Darmstadt, Germany; Merck Schuchardt MS Info 88-1, cat. no. 818372). It is a crystalline solid that is soluble in water and low MW alcohols, and it was originally recommended for the selective oxidation of organic sulfides to sulfoxides and sulfones (19,20).

8. TS- and TSI-gels show high stability in 0.2 M sodium acetate, pH 5.0, at 4°C, because no decrease in reactivity can be detected after long storage periods under these conditions (6,8).

9. The antibacterial properties of alkyl thiolsulfinates are well known and we have never observed microbial growth in these gel suspensions.

10. This potassium phosphate buffer is the most suitable for this P-galactosidase because sodium ions inactive the enzyme, and also because of the presence of magnesium ions, which stabilizes the enzyme.

11. Use gentle suction to remove liquid until the moment when the gel forms a firm cake and then stop suction; the gel is then called suction-dried gel.

12. Plastic reservoir with a bottom membrane and top and bottom caps.

13. This E. coli P-galactosidase is selected because of its high cysteine content; it should therefore be possible to use its nonessential thiol groups for its immobilization.

14. Sodium chloride is included in the washing buffer to elute nonspecifically bound proteins.

15. The percentage of gel-bound activity depends on the amount of protein added, ranging from 70% to 80% for the low- and intermediate-load derivatives, to 41% for the high-load derivative. A control gel (with its thiol-reactive groups previously blocked with glutathione) does not retain the enzyme.

16. In 3.0 mL of 8 mM glutathione solution there is a tenfold excess of glutathione with respect to the number of active groups present on the activated gel. Other thiol compounds, like mercaptopropionic acid and mercaptoethanol (in activity buffer) can be used for blocking the excess of active groups. Because of its low pKa value, cysteamine (pKa = 8.3) is the only thiol-compound assessed that has an eluting effect. (After the blocking treatment with cysteamine under the conditions described, nearly 30% of the bound enzyme is released.)

17. More than 90% of the total immobilized activity is eluted, proving that the matrix-enzyme bonds formed are of disulfide type.

18. Prior to its use, this commercial lactase preparation (Maxilact LX-5000) is diluted 10-fold with 20 mM phosphate buffer, pH 7.0, and gel filtered through Sephadex G-25.

19. Under these conditions, the ratio |imoles SH groups of reductant/mg of protein is 3.4.

20. Achieving a ratio of |imoles SH groups of reducing agent/mg of protein of 0.5.

21. The reduction of this lactase with both reductants allowed a threefold increase in its initial content of SH groups. Nearly sevenfold less |imoles of SH groups/mg of protein were needed to perform the reduction of K. lactis P-galactosidase with thiopropyl-agarose than the SH amount required for the reduction with DTT.

22. After the reduction process, the remaining content of SH groups of the solidphase reducing agent was quantified; nearly 60% of the initial SH groups remained.

23. The reduction process dramatically improved the immobilization yield onto thiol-reactive supports, from 0% for the native enzyme up to 90% for the reduced enzyme.

24. Using an acrylic resin as support, like Toyopearl HW-65F, the expressed P-ga-lactosidase activity is strongly dependent on the salt concentration present during the immobilization process, increasing from 32% in absence of salt to 60% in the presence of 0.3 M K2SO4 (Table 2).

25. The immobilization process requires incubation periods of at least 8 hours for quantitative binding of the reduced enzyme to TS- and TSI-supports (see Fig. 5).

26. After overnight recirculation, the immobilization yield for the reduced enzyme is 69%.

27. The blocking treatment markedly improves the thermal stability at 37°C (optimum temperature for the hydrolysis of lactose) of P-galactosidase derivatives (Table 3).

Table 2

Immobilization Yields of Reduced ß-Galactosidase Onto Thiol-Reactive Supports

Table 2

Immobilization Yields of Reduced ß-Galactosidase Onto Thiol-Reactive Supports

Thiol-reactive support

Immobilization yield (%)a

Expressed activity (%)b

TS-agarose

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