EP0000486A1 - Method for preparing stabilized carrier-bound proteins - Google Patents

Abstract

Stabilized carrier-bound proteins are obtained by reacting a protein in aqueous solution with a water-insoluble, mercapto group-free carrier material, which contains groups that bind to proteins, and by subsequent exposure to hydrogen sulfide or preferably aliphatic compounds with a molecular weight below 5000, preferably below 2000, with at least a mercapto group and at least one further mercapto, hydroxy or primary or secondary amino group. Preferably, a bead-shaped carrier material with a bead diameter between 5 and 1000 µm based on a cross-linked polyacrylamide with oxirane groups or cyanbrominated cellulose, agarose or dextrans with an enzyme, e.g. Penicillin acylase implemented. Then at least 0.03% by weight of free mercapto groups are added to the carrier material. The products are characterized by reduced degradation of biological activity with multiple reuse of the bound protein.

Description

The invention relates to a process for the production of stabilized carrier-bound proteins. In processes which are based on the biospecific interaction between a protein and a corresponding substrate, the proteins are advantageously used in the form of a material bound to a solid carrier. The protein can thereby be separated from the treated substrate solution by simple filtration. Carrier-bound proteins are important in affinity chromatography and especially in carrying out enzymatic processes, the protein bound by the carrier being an enzyme.

Compared to the free proteins in aqueous solution, carrier-bound proteins are generally characterized by increased stability against inactivation through a pH shift, high temperatures or autoxidation. Nevertheless, the biospecific activity, for example in the case of enzymes the enzymatic activity, drops somewhat with repeated use of the carrier-bound protein at each use level. It is known that the Loss of activity of proteins can be stopped or reversed if they are treated with mercaptans (see dissertation Jan Carlsson, Uppsala 1974, p. 8, abstract). The effect of the mercaptans is attributed to the fact that they break down disulfide bridges within the protein molecule which have arisen from thiol groups by autoxidation. This auto-oxidation is considered to be the cause of the reversible loss of activity.

However, in most cases it is not desirable. To maintain the activity of the bound protein of the substrate solution, add a mercaptan because the substrate solution is contaminated thereby and an additional, complex cleaning step is required. The separate reactivation of the carrier-bound protein between two applications is also not a satisfactory solution because of the additional work involved. In addition, most mercaptans are toxic and smelly.

The object of the present invention was to produce carrier-bound proteins with increased stability against gradual loss of activity in aqueous solution by reacting the protein with a water-insoluble carrier material. The object is achieved according to the invention in that hydrogen sulfide or a compound having a molecular weight below 5000, preferably below 2000, with at least one mercapto group and at least one further mercapto group, Hydroxy or primary or secondary amino group can act.

Since experts believed that the stabilizing effect of mercaptans is based on their reaction with disulfide bridges, it was not expected. that they would be effective even if they were bound to the support material and were therefore unable to get close to the disulfide bridges. Surprisingly, the bound mercapto groups nevertheless achieve considerable stabilization. The following table uses the example of a carrier-bound penicillin acylase to compare the loss of activity with multiple reuse with and without mercapto groups on the carrier. In this case, the mercapto groups were introduced by binding dithioerythritol to an enzyme carrier containing epoxide groups. The carrier material was a crosslinked acrylamide polymer with epoxy groups, which had first been reacted with penicillin acylase and then optionally with 50 or 500 mg of dithioerythritol per gram of the moist carrier material. The activity of the preparation against 6-amino-penicillinic acid was determined in a repeated reaction of 1 to 2 g of the preparation, each with 5% substrate in 200 ml of substrate solution (0.01 M phosphate buffer, pH 7.5) at 37 ° C. by automatic titration Determined 1.0 N NaOH. The initial activity when used for the first time was designated as 100%.

The stabilization achieved according to the invention is based on the binding of hydrogen sulfide or a compound containing at least one mercapto group and at least one further mercapto, hydroxyl or primary or secondary amino group to the binding-active groups of the carrier material. The mercapto, hydroxyl or amino groups of the compounds mentioned, just like the corresponding groups of the proteins to be bound, react with the binding-active groups of the support material. Since the compounds have a molecular weight below 5000, they are still able to react with such binding-active groups of the carrier material due to their small molecular size, which are inaccessible to protein molecules due to steric hindrance. The binding-active groups react more easily with hydrogen sulfide or with mercapto, hydroxy and amino groups than with water, so that it is possible to react both the binding-active groups with the protein and the subsequent reaction with the mercapto and optionally hydroxy or To carry out amino group-containing compound in the aqueous phase. Since the binding-active groups are nevertheless gradually decomposed by water, there should not be an unnecessarily long period of time between the first and the second implementation stages of the process according to the invention.

The desired stabilizing effect occurs when at least one mercapto group per bound molecule is retained in free form and the further mercapto, hydroxyl or amino group reacts with the binding-active group of the carrier. Since the carrier consists of an insoluble solid matrix and has a correspondingly low molecular mobility, the compound containing mercapto groups is largely bound to the carrier only with one of its functional groups. If this group is a hydroxyl or amino group, a mercapto group is definitely retained. When using a compound with only one mercapto group and one or more amino or hydroxy groups, the mercapto group can react with the binding-active group of the support, so that only amino or hydroxyl groups remain. In this case, the bound Molecule does not contribute to stabilization. However, since some of the molecules do not react with the mercapto group but with an amino or hydroxyl group, there are enough stabilizing mercapto groups left on average.

The amount of the mercapto group-containing compound is generally such that at least 0.3% by weight, based on the wet weight of the carrier enzyme product, is bound to free SH groups. Hydrogen sulfide and compounds with 2 or more mercapto groups contain two (or more) hydrogen atoms bonded to sulfur, so that at least one free mercapto group remains after their binding to the support. Compounds with two or more SH groups are particularly preferred. These include dithioethylene glycol, dithiopropylene glycol or dithiobutylene glycol, 1,12-dimerkaptododecane, di-mercaptoacetic ester of ethylene glycol, dodecanediol, neopentyl glycol or 2,2'-bis (4-hydroxy-cyclohexyl) propane, trimethylolpropanoate, trimethylolpropanoate, trimethylolpropanoate, trimethylolpropanoate, trimethylolpropanoate mercaptoacetate, tetra-mercaptopropionate. -tetra-mercaptocaproic acid ester or -tetra-mercapto and ecanoic acid ester as well as compounds with further functional groups such as dithioerythritol. As compounds with one amino and one mercapto group, mercaptoethylamine and cysteine may be mentioned. Compounds with only one mercapto and one or more hydroxyl groups are the least. preferred since the mercapto groups generally react more easily than the hydroxyl groups with the binding-active groups of the support. Examples this type of compound is monothioethylene glycol and monothiopropylene glycol.

A large number of water-insoluble carrier materials with groups which are active against binding to proteins are known. An overview of such carriers and their active groups can be found in 0. Zaborsky, "Immobilized Enzymes", Cleveland 1973. The most important carrier materials are synthetic crosslinked polymers based on acrylamide, maleic anhydride, methacrylic acid, styrene or polypeptides. Natural carriers are mentioned Carbohydrates such as agarose, cellulose, cross-linked dextran or starch are mentioned. Glass and other inorganic substances can also be used as carriers. The binding active groups include, for example, activated support Nitroarylfluorid _-, Silylchlorid-, carboxylic anhydride, carbonyl, carbonate, imido, - isothiocyanate, (nitro) Phenylester-, Acylazid-, aryl diazonium or cyanuric chloride groups. Methods for introducing these groups into the carrier materials are specified in detail in the book mentioned and in the literature references cited here. The number of binding-active groups should be large enough to bind at least 10 mg, preferably 100 mg or more protein per gram of carrier material.

The stabilizing effect is particularly evident in those carriers which contain oxirane or amidocarbonate groups as binding-active groups. Oxirane groups can already be tion of a crosslinked acrylamide polymer, preferably a bead polymer produced by reverse suspension polymerization, by incorporating comonomers containing glycidyl groups, such as glycidyl acrylate or methacrylate. Glycidyl groups can be introduced into carrier bodies with a carbohydrate structure, such as cellulose, agarose or dextran, for example by reaction with epichlorohydrin or 1,4-bis (2,3-epoxypropyl) butane. By reacting the last-mentioned carrier bodies with cyanogen bromide, imidocarbonate groups are obtained. Pearl-shaped carriers with pearl diameters of 5 to 5000 µm are particularly advantageous because of their easy handling and their flow permeability.

The nature of the protein to be bound depends on the intended use of the process product. For the purposes of affinity chromatography, a protein is bound to the support, which can interact in a biospecific manner with a component of a substrate mixture to be separated. This component is temporarily held on the carrier-bound protein. Enzymes are preferably bound to the carrier, such as, for example, penicillin acylase, ribonuclease, amylase or catalase. Penicillin acylase bound and stabilized in the sense of the invention is of particular technical importance because this is the only way to economically reuse this valuable enzyme on an industrial scale.

Some enzymes are sensitive to hydrogen sulfide or mercaptans; they are then generally not suitable for the process of the invention. Enzymes which are already stabilized or reactivated by hydrogen sulfide or free mercaptans can be converted into enzyme products of increased stability by the process of the invention.

example 1 a) Production of oxirane acrylic resin beads

• 1740 g n-Heptan
• 1100 g Perchloräthylen
• 2 g Polymerisat der Zusammensetzung
  • 95 % n-Butylmethacrylat
  • 5 % 2-Trimethylammoniumäthylmethacrylat-chlorid
  
  vorgelegt. Unter Einleitung von Stickstoff wird bei ca. 55°C eine Mischung aus
• 791 g Formamid
• 60 g Methacrylamid
• 60 g Allylglycidäther
• 60 g Glycidylmethacrylat
• 120 g Methylen-bis-methacrylamid
• 6 g Benzoylperoxid

zugegeben und durch Rühren in der organischen Phase verteilt. Die Polymerisation wird durch Zugabe von 6 g Dimethylanilin beschleunigt. Nach 10-stündiger Polymerisation wird auf Raumtemperatur abgekühlt, die Perlen absetzen gelassen und die überstehende flüssige Phase abdekantiert. Die Perlen werden durch Zugabe von ca. 2000 ml Aceton entquollen, abgesaugt und zweimal mit Aceton nachgewaschen. Anschließend werden sie über Nacht bei Raumtemperatur im Vakuum getrocknet. Ausbeute: ca. 90 % feine, leicht gelbl. Perlen.In a round bottom flask (6000 ml) equipped with a thermometer, impeller stirrer, reflux condenser and N ₂ inlet pipe

• 1740 g of n-heptane
• 1100 g perchlorethylene
• 2 g of polymer of the composition
  • 95% n-butyl methacrylate
  • 5% 2-trimethylammoniumethyl methacrylate chloride
  
  submitted. A mixture is formed at about 55 ° C. with the introduction of nitrogen
• 791 g formamide
• 60 g methacrylamide
• 60 g allyl glycidyl ether
• 60 g glycidyl methacrylate
• 120 g methylene bis methacrylamide
• 6 g benzoyl peroxide

added and distributed in the organic phase by stirring. The polymerization is accelerated by adding 6 g of dimethylaniline. After polymerization for 10 hours, the mixture is cooled to room temperature, the beads are allowed to settle and the supernatant liquid phase is decanted off. The beads are swollen by adding about 2000 ml of acetone, suction filtered and washed twice with acetone. Then they will dried overnight at room temperature in a vacuum. Yield: approx. 90% fine, slightly yellow. Pearls.

b) immobilization of penicillin acylase

1.2 g penicillin acylase (E. coli, specific activity 12.1 U / mg) are dissolved in 40 ml 0.1 M phosphate buffer (pH 8, 0.02% NaN ₃ ) and added to 10 g of the product (1a) given. The mixture is left to stand at 23 ° C. for 72 hours and then washed three times with 1000 ml of 1 M aqueous NaCl solution and twice with 0.05 M phosphate buffer (pH 7.5; 0.02% NaN ₃ ).

Yield: 37 g of moist product (after suction on glass frit). Enzymatic activity: 237 U / g wet product.

c). Activity determination

2 g penicillin-G (potassium salt) are dissolved in 90 ml phosphate buffer (pH 7.5; 0.05 M) and the volume is brought to 100 ml with this buffer.

20 ml of this substrate solution and 250 mg (wet weight!) Of the product (1b) are stirred in a titration stand (TTA 3, Radiometer, Copenhagen) at 37 ° C thermostatted. The phenylacetic acid released by hydrolysis is automatically titrated at pH 7.8 with 1 N NaOH from a car burette (ABU 12, Radiometer, Copenhagen), controlled via pH meter and titrator (type 11, Radiometer, Copenhagen). Simultaneously, the NaOH consumption is registered with the help of a recorder (Titrigraph SBR 2c, Radiometer, Copenhagen).

The activity is determined graphically from the linear starting area of the sales curve; 1 enzyme unit (U) corresponds to 1 µmol of split substrate per minute.

d) Loss of activity after repeated use at 95%

Sales of substrate solutions with a concentration of 5%

2.0 g of the product (1b) and 20 ml of a 5% substrate solution (5% penicillin-GK in 0.01 M phosphate buffer, pH 7.5 containing 0.02% NaN ₃ ) are at pH 7.5 and 37 ° C stirred up to a substrate conversion of 95-98%. Apparatus and. Registration of the turnover curve as described under (1c).

Then the enzyme beads are sucked off on a glass frit and used again against 20 ml of substrate solution. This procedure is repeated 20 times in a row.

Loss of activity after 20 uses: 57%.

Example 2

. 10 ml of an aqueous solution of dithioglycol (2% adjusted to pH 7.5 with NaOH) are added to 2 g of the carrier-bound penicillin acylase according to Example 1 (b). The mixture is left to stand at 37 ° C. for 18 hours and washed with water (ten times 100 ml each time) and twice with 0.05 M phosphate buffer (pH 7.5).

Yield: 1.95 g wet product.

The product is tested as in Example 1 (a).

Loss of activity after 20 repeated uses dung: 42%.

Example 3

50 mg of 1,4-dithioerythritol are dissolved in 1 ml of H ₂ O and the pH is adjusted to 7.5 by adding NaOH. The solution is added to 2 g of carrier-bound penicillin acylase according to Example 1 (b) and the mixture is left to stand at 23 ° C. for 48 hours. Then it is washed with water (five times) and buffer (twice).

Yield: 1.98 g

Loss of activity after 20 repeated uses performed as in Example 1 (d): 35%.

Example 4

500 mg of dithioerythritol are dissolved in 5 ml of water and a pH of 7.8 is set by adding NaOH. This solution becomes 2 g of the carrier-bound. Penicillin acylase according to Example 1 (b) was added and the mixture was left to stand at 23 ° C. for 48 h.

Then it is washed with water (five times) and buffer (twice).

Loss of activity after 20 repeated uses, carried out as in Example 1 (d): 11%.

Claims (9)

1. Process for the preparation of stabilized carrier-bound proteins by reacting a protein in aqueous solution with a water-insoluble carrier material which contains groups which are active against proteins,
characterized,
that one acts on the protein bound to a mercapto group-free carrier hydrogen sulfide or a compound with a molecular weight below 5000, preferably below 2000, with at least one mercapto group and at least one further mercapto, hydroxyl or primary or secondary amino group. 2. Ver ears according to claim 1, characterized in that a carrier material is used which contains oxirane or imidocarbonate groups as groups which are active against binding to proteins. 3. The method according to claim 2, characterized in that a carrier material based on a polyacrylamide or carbohydrate derivatives is used. 4. The method according to claims 2 and 3, characterized in that. a pearl-shaped carrier material with a pearl diameter between 5 and 1000 μm on the basis of a cross-linked poly acrylamides with oxirane groups is used as groups reactive towards proteins. 5. Process according to claims 2 and 3, characterized in that cyan brominated carbohydrates, for example cellulose, cross-linked dextrans or agarose, are used as the carrier material. 6. Process according to claims 1 to 5, characterized in that an aliphatic compound having 2 to 8 carbon atoms is used as the compound having at least one mercapto group. 7. The method according to claims 1 to 6, characterized in that the bound protein is an enzyme. 8. The method according to claim 7, characterized in that the bound enzyme is penicillin acylase. 9. The method according to claims 1 to 8, characterized in that at least 0.03 wt .-% (based on the wet weight of the end product) of free mercapto groups are added to the carrier material.