Method for Immobilizing Dissolved Proteins

Support: Macroporous highly crosslinked styrene/divinylbenzene beads having an inner surface area of about 200 m.sup.2 /g and an average pore diameter of 40 nm.

Enzyme: Glucose isomerase, liquid concentrate from a culture of Streptomyces albus.

Activity: 1 g of enzyme converts D-glucose from a 0.1 molar solution at pH 7 and 70.degree. C. to 5 g of D-fructose in 60 minutes.

Coupling: First the enzyme is adsorbed onto the macroporous support. To this end, 10 g of support and 10 g of enzyme in 50 ml of saline solution containing 12% of sodium sulfate, 5% of magnesium sulfate, and 0.02% of cobalt sulfate are rolled at room temperature (23.degree. C.) on a roller table. The ionic strength of the aqueous solution is 4.18 moles/liter. The pH value was adjusted to 7.0. After 20 hours, 0.5 g of glutaraldehyde is added and rolling is continued. The adsorbed enzyme is crosslinked and retained in the pores by this process.

Filtration by suction and washing follow 2 hours later. A comparative activity determination shows an activity of 45% in the filtrate and of 37% in the support.

Activity Yield: 37%.

Use: The immobilized glucose isomerase is used as packing in a column reactor. At a temperature of 60.degree. C. and a throughput rate of seven times the fixed-bed volume per hour, a 40% glucose solution of pH 7.5 is isomerized to fructose with 45% conversion.

Support: Crosslinked polyacrylic ester; average pore diameter, 25 nm; inner surface area, 140 m.sup.2 /g; beads from 0.3 to 1 mm in diameter.

Enzyme: Aspergillus oryzae lactase, powdered concentrate. Activity: 30,000 U/g.

Coupling: 10 g of support are shaken with 1 g of enzyme preparation in 40 ml of saline solution at 35.degree. C. for 8 hours. The saline solution contains 24% of potassium chloride and was adjusted to pH 5.0. The ionic strength of the aqueous solution is 3.21 moles/liter. 0.1% of lactose was further added for stabilization of the enzyme. After cooling, shaking is continued for another 2 hours at room temperature with addition of 0.5% of glutaraldehyde. This is followed by filtration by suction and washing. The lactase activities of support and filtrate are then compared. The support is found to have 34% of the initial activity, the filtrate, 11%.

Activity yield: 34%.

Use: The immobilized Aspergillus lactase is used as packing in a column reactor. At a temperature of 35.degree. C., a lactose solution of pH 4.5 is hydrolyzed at a throughput rate of 40 fixed-bed volumes per hour with over 90% conversion. After 60 days, no loss of activity is observable.

Support: Macroporous, highly crosslinked bead polymer comprising methacrylamide/methylenebismethacrylamide having free epoxy groups (1.2% oxirane oxygen) and 2.2% of adhering isopropenyl groups which are concentrated on the inner surfaces of the pores. Pore volume, 3.4 ml/g. Average pore diameter, 20 nm. The preparation of this support is described in Example 2 of German patent publication 27 22 751.

Coupling of the adsorbed enzyme here is effected covalently by reaction with the epoxy groups simultaneously with adsorption of the enzyme under the influence of the high salt concentration.

Enzyme: Yeast lactase from Saccharomyces (Kluyveromyces) lactis, liquid preparation 5,000 neutral lactase units (NLU).

Coupling: 10 g of support are shaken at room temperature (23.degree. C.) with 10 g of enzyme in 80 g of saline solution. The latter contains 16% of dibasic potassium phosphate, 7.9% of monobasic potassium phosphate and, for stabilization of the enzyme, 20 ppm of MnCl.sub.2. 4H.sub.2 O. The solution has an ionic strength of 3.34 moles/liter. After 72 hours, filtration by suction and washing are carried out and the lactase activity of the filtrate is compared with that of the support. The support is found to have 55% of the initial activity and the filtrate 14%.

At 55%, the activity yield is very high in the case of this sensitive enzyme.

Use: The coupled yeast lactase is used as packing in a column reactor. At a temperature of 7.degree. C., skim milk with 0.3% fat is passed through it for 20 days at a flow rate of 55 fixed-bed volumes per hour. The lactose contained in the milk is hydrolyzed to glucose and galactose, at first with 65% conversion, and after 20 days with 50% conversion.

Support: Macroporous highly crosslinked bead polymer comprising methacrylamide/methylenebismethacrylamide having free epoxy groups (1.2% oxirane oxygen) and 2.2% adhering isopropenyl groups which are concentrated on the inner surfaces of the pores. Pore volume, 3.4 ml/g. Average pore diameter, 20 nm. The preparating of this support is described in Example 2 of U.S. Pat. No. 4,208,309.

Enzyme: Powdered aminoacylase concentrate from an Aspergillus strain.

Activity: 23,000 U/g. Substrate: Acetyl-D, L-methionine.

Coupling: 10 g of support are shaken at 35.degree. C. with 20 g of enzyme preparation in 80 ml of saline solution. The latter contains 14.2% of sodium sulfate and 24 ppm of CoCl.sub.2.6H.sub.2 O and is adjusted to pH 7.0. Its ionic strength is 3 moles/liter, disregarding the salt content of the enzyme preparation.

After 8 hours, the support is filtered by suction and washed. The support is found to have 61% of the initial activity and the filtrate, 1%. The activity yield thus is 61%.

3.5 g portions of moist support material according to Table III are washed five times, each time with five times their volume of desalinated water, and then suction filtered on a porous glass plate. Then the support material is shaken at about 21.degree. C. for 2 hours with 6.8 ml of an enzyme solution containing 676 international units (IU) of penicillinamidase from E. coli in 0.5 M of potassium phosphate buffer (pH 7.5, with 0.1% NaN.sub.3). The ionic strength of the buffer solution is 1.5 moles/liter. After the addition of 0.136 ml of a 25% aqueous glutaraldehyde solution which had been stabilized with an ion exchange resin (Amberlite A 21), shaking is continued for 2 hours. The loaded support material is then placed on a porous glass filter and washed three times with 1 M NaOH and twice with 0.05 M sodium phosphate buffer (pH 7.5, with 0.1% NaN.sub.3).

The enzymatic activity was determined by alkalimetric titration at pH 7.5 using penicillin G K (crude) as a substrate. For this purpose, 20 ml each of the 2% substrate solution in 0.05 M sodium phosphate solution at pH 7.5 were used and automatically titrated at 37.degree. C. with 0.5 M sodium hydroxide solution. The results are presented in Table III.

3.5 g (moist weight) of "Phenyl-Sepharose" are pretreated as in Example 1. Then 150 mg of trypsin dissolved in 6.8 ml of 0.5M potassium phosphate buffer (pH 7.5, with 0.1% NaN.sub.3) are added, followed by shaking at 23.degree. C. for 2 hours. The ionic strength is 1.5 moles/liter. Immobilization of the adsorbed enzyme and further treatment are carried out as in Example 1.

The enzymatic activity was determined using casein and N-benzoyl-1-arginine ethyl ester hydrochloride (BAEE) as substrates.

1 g portions of Aspergillus oryzae lactase "Lactase Preparation 2214 C Conc." having a strength of 30,000 U/g are dissolved in 40 ml portions of a 0.7 M Na.sub.2 SO.sub.4 solution having an ionic strength of 2.1 moles/liter at pH 5.5. Portions of such solutions are then mixed in each case with 10 g of one of the carrier resins listed in Table IV and shaken for 20 hours at room temperature. 0.8 ml of 25% aqueous glutaraldehyde solution is then added and shaking is continued for 2 hours at room temperature. The preparations are separated by filtration and washed and the activity of the immobilized enzyme and of the enzyme found in the filtrate is determined and expressed in percent of the initial activity. The results are presented in Table IV.

1 g of lactase from Aspergillus oryzae ("Lactase Preparation 2214 C Conc.") having a strength of 30,000 U/g is dissolved in 40 ml of a 0.7 m Na.sub.2 SO.sub.4 solution having a ionic strength of 2.1 moles/liter al pH 5.5. This solution is then mixed with 10 g of the carrier (b) out of Table IV. At the same time 0.8 ml of 25% aqueous glutaraldehyde solution is added and this suspension is shaken for 20 hours at room temperature.

Crosslinking happens here during adsorption.

The preparation ia separated by filtration and washed and the activity of the immobilized enzyme and of the enzyme found in the filtrate is determined as 25% respectively 4%.

Example 8 is repeated, but the addition of the aqueous glutaraldehyde to the enzyme solution occurs 20 minutes before the carrier is added.

Crosslinking happens here before adsorption.

The activity of the immobilized enzyme and of the enzyme found in the filtrate is determined as 17%, respectively 0%.