Method for immobilizing dissolved proteins

Methods for adsorbing a protein, for example an enzyme, onto an insoluble, solid, macroporous, small-particle support by washing said support with an aqueous solution of the protein containing an electrolyte in an ionic strength of at least 0.15 mole/liter and crosslinking said protein, before, during, or after such adsorption, with a coupling component present in aqueous electrolyte-containing solution.

EXAMPLE 1 
Immobilized glucose isomerase 
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. 
EXAMPLE 2 
Immobilized Aspergillus oryzae lactase 
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. 
EXAMPLE 3 
Immobilized yeast lactase 
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. 
EXAMPLE 4 
Immobilized aminoacylase 
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%. 
EXAMPLE 5 
Binding of penicillinamidase to various supports 
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. 
TABLE III 
__________________________________________________________________________ 
Activity of Immobilized 
penicillinamidase 
Support Material U/g Activity 
Chemical composition 
Trade name moist weight 
Yield 
__________________________________________________________________________ 
(a) 
Crosslinked agarose 
("Sepharose-CL-4B") 
96 58 
(b) 
Octylated cross- 
("Octyl-Sepharose- 
98 53 
linked agarose 
CL-4B") 
(c) 
Phenylated cross- 
("Phenyl-Sepharose 
168 91 
linked agarose 
CL-4B") 
(d) 
Cross linked agarose 
("DEAE-Sepharose- 
84 61 
substituted with 
CL-6B") 
diethylaminoethyl 
groups (cataionic) 
(e) 
Carboxymethylated 
("CM-Sepharose- 
94 48 
crosslinked agarose 
CL-6B") 
(anionic) 
(f) 
Phenoxyacetylcellulose 77 59 
(g) 
Oxirane-polyacrylamide resin 
172 85 
(according to U.S. 4,208,309, 
(Example 1), reacted with benzyl thiol 
(h) 
Polymethacrylimide ("Rohacell WF") 
70 45 
foam, ground 
(i) 
Crosslinked polymethyl methacrylate/ 
35 25 
glycol dimethacrylate copolymer**, 
weight ratio 90:10 
(j) 
Crosslinked polymethyl methacrylate/ 
55 37 
glycol dimethacrylate copolymer**, 
weight ratio 80:20 
(k) 
Crosslinked polymethyl methacrylate/ 
43 33 
glycol dimethacrylate copolymer**, 
weight ratio 60:40 
(l) 
Crosslinked polystyrene 
57 42 
with 10% divinylbenzene 
(m) 
Porous glass, ("Controlled Pore Glass 10") 
105 60 
pore volume 0.75 cm.sup.3 /g, 
average pore diameter 17 nm, 
inner surface area 107 m.sup.2 /g 
__________________________________________________________________________ 
**Method of preparation: A solution of 1 part by weight of polyvinyl 
alcohol in 320 parts of water is heated to 50.degree. C. in a stirred 
vessel and a mixture of 100 parts of monomers (methylmethacrylate and 
glycol dimethacrylate or styrene and divinylbenzene, respectively), 60 
parts of nheptane, and 1.4 parts of dibenzoyl peroxide is dispersed 
therein as droplets with stirring. During the 4hour polymerization time, 
the temperature is held by cooling to a maximum of 75.degree. C. After 
that, the solvent is distilled off over 1 hour at 36.degree. C. The 
polymer beads formed are separated by filtration after cooling. 
EXAMPLE 6 
Binding of trypsin to "Phenyl-Sepharose" 
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. 
______________________________________ 
Results: Using casein 
8.3 U/g moist weight 
Using BAEE 311 U/g moist weight 
______________________________________ 
EXAMPLE 7 
Production of support-bound lactase preparations 
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. 
TABLE IV 
__________________________________________________________________________ 
Activity 
Immobilized 
Residual 
lactase activity 
Activity 
in 
yield filtrate 
Carrier Material (%) (%) 
__________________________________________________________________________ 
(a) 
Weakly basic ion 17.5 0 
exchanger based on 
styrene/divinyl- 
benzene ("Amberlite IRA 93") 
(b) 
Macroporous adsorber 29.5 18 
resin comprising poly- 
acrylate basis ("Amberlite XAD 7") 
(c) 
Macroporous phenol- 24.0 5 
formaldehyde adsorber 
resin, weakly basic ("Duolite S 561") 
(d) 
Macroporous phenol- 26.0 4 
formaldehyde adsorber 
resin, weakly basic ("Duolite S 587") 
(e) 
Macroporous phenol- 22.0 6 
formaldehyde adsorber 
resin, nonionic ("Duolite S 761") 
(f) 
Macroporous glass, 42.4 8 
pore volume 
0.75 cm.sup.3 /g, average 
pore diameter 17 nm, 
inner surface area 
107 m.sup.2 /g ("Controlled Pore Glass CPG 10") 
(g) 
Polymethacrylimide 31.0 0 
foam, ground ("Rohacell") 
(h) 
Hydroxyl apatite 16.5 4 
(basic calcium 
phosphate), ground 
__________________________________________________________________________ 
EXAMPLE 8 
Production of support-bound lactase preparation:Crosslinker is added at the 
beginning of adsorption 
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 9 
Production of support-bound lactase preparation:Crosslinker is added before 
adsorption 
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%.