3.3.1. Activity Determination
The activity determination is performed as in Subheading 3.2.1.
3.3.2. Protein Determination
1. The protein content of the enzyme solutions is determinated with Coomassie Plus reagent.
2. Immobilized protein is estimated as the difference between the amount of protein added to the gel and that recovered in the pooled supernatant and washing fractions. It is also determined by total amino acid analysis after extensive drying over phosphorus pentoxide in a dessicator and hydrolysis in 6 MHCl for 24 h at 110°C.
3.3.3. Enzyme Reduction With a Soluble Reducing Agent
1. Incubate aliquots of enzyme (60 mg/mL, 1400 enzyme units [EU]/mL) at 22°C with DTT 200 mM dissolved in 20 mM potassium phosphate, pH 8.0 (see Notes 18 and 19).
2. After 30 min, remove the excess of DTT by gel-filtration.
3. Determine protein SH content (21).
3.3.4. Enzyme Reduction With a Solid-Phase Reducing Agent
Using thiol-agarose containing 1000 ^moles SH groups per gram of dried gel, prepared as described in Subheading 3.1.1.
1. Incubate aliquots of enzyme (8 mg/mL, 240 EU/mL) at 22°C with 1.0 g suction-dried thiopropyl-agarose.
2. After 2 h, remove the reducing agent by filtration on a sintered glass filter.
3. Titrate the thiol content of the reduced enzyme (21) (see Notes 20-22).
3.3.5. Preparation of Immobilized Enzyme Following a Batch Procedure
1. Follow the protocol described in Subheading 3.2.3. using reduced enzyme (see Notes 23-25).
3.3.6. Preparation of Immobilized Enzyme Following Column Procedure
1. Equilibrate TS-agarose or TSI-agarose as in Subheading 3.2.3.
2. Pack 5 mL of this gel into a plexiglass column.
3. Recirculate for 16 h at 22°C, reduced and gel filtered enzyme in 0.1 Mphosphate buffer, pH 7.0, at a flow rate of 10 mL/h.
4. Wash the column sequentially with 0.1 M phosphate buffer, pH 7.0, with and without 0.5 M NaCl.
5. Measure P-galactosidase activity in the recirculated and washing fractions, and in the gel-derivative obtained (see Note 26).
3.3.7. Blocking Excess Active Groups
1. Incubate for 30 min, 300 mg of suction-dried gel derivatives with 3.0 mL of fresh 8 mM glutathione solution.
2. After incubation, filter, wash, and dilute to 3.0 mL with activity buffer (see Note 27).
3.3.8. Enzyme Elution
1. Follow the protocol described in Subheading 3.2.5.
2. After filtration, determine protein content and activity in the filtrates (see Note 28).
1. Incubate at 22°C and 37°C, aliquots of free and immobilized enzyme suspensions with 5% lactose saline solution, whey, whey permeates and skimmed milk (ratio 1/10 [v/v]).
2. Glucose formation is followed by an enzymatic method (see Note 29).
3.3.10. Lactose Hydrolysis in Column
1. A column with 5 mL of packed gel is fed with the same lactose solutions specified above, using a flow rate between 6.0 and 10.0 mL/h.
2. Determine the glucose formed (see Note 30).
3.3.1. Reuse of the Immobilized Enzyme
1. Incubate with whey at 22°C for 2.5 h an aliquot of the TSI-agarose-P-galactosi-dase derivative.
2. Determine the amount of glucose formed.
3. Wash the derivative with activity buffer.
4. Use the washed derivative for a second time in the same way as in the first time.
5. This protocol is carried out four times (see Note 31).
After release of bound material from the gel derivative by reductive cleavage and thorough washing, reactivation is performed according to the two-step procedure described for TSI-gel synthesis or the one-step procedure described for TS-gel (see Notes 32 and 33).
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