JPH01179688A - Novel production of modified enzyme and novel modified enzyme - Google Patents

Abstract

PURPOSE:To synthesize a modified enzyme and an immobilized enzyme, by linking an azo compound having polymerization initiating ability to the amino group on the surface of an enzyme by covalent bond using a condensation agent and subjecting a vinyl monomer to graft polymerization onto the compound. CONSTITUTION:A azo compound having polymerization initiating ability to the amino group on the surface of an enzyme is linked to the surface of an enzyme by covalent bond and any of various vinyl monomers is subjected to graft polymerization onto the compound to give a modified enzyme, namely, an enzyme.vinyl polymer hybrid or an immobilized enzyme of new type. An esterase such as choline esterase or cholesterol esterase may be cited as the enzyme. Specific examples of the azo compound having polymerization initiating ability to the amino group on the surface of an enzyme are 4,4'-azobis(4- cyanovaleric acid), 2,2'-azobis(4-carboxy-2-methylbutyronitrile), etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel method for producing a modified enzyme, and a novel modified enzyme (enzyme/vinyl polymer hybrid or immobilized enzyme) obtained thereby.

[Background of the invention]

Modified enzymes include chemically modified water-soluble modified enzymes and so-called immobilized enzymes immobilized on insoluble carriers.

In recent years, a lot of research has been carried out on so-called protein hybrids, in which synthetic polymers are bonded to proteins such as enzymes. It is attracting a lot of attention because it not only allows for efficient reaction, but also allows for condensation reactions, which are the reverse reactions to hydrolysis. However, the only method currently generally used to produce this type of modified enzyme is to synthesize a polymer substance as a modifier in advance and combine it with the enzyme to synthesize a hybrid. At present, the polymeric substances used as such have so far been limited to polyethylene glycol.

On the other hand, various types of so-called immobilized enzymes have been used industrially in a wide range of fields, but the conventionally known enzyme immobilization and its methods can be roughly divided into It was limited to the following three types.

l) Inclusion method: A water-soluble monomer and a water-soluble cross-linking agent are added to an enzyme aqueous solution to polymerize and gel, thereby sealing the enzyme in the Kel matrix space.

2) Cross-linking method: A method in which an enzyme solution is reacted with a cross-linking agent (generally a low-molecular compound) to form bonds between enzyme molecules, resulting in an enzyme cross-linked product.

3) t4-body bonding method: A method in which an enzyme and a polymeric material (carrier) on which the enzyme is to be supported are bonded by covalent bonding, ionic bonding, etc. in accordance with the method of polymeric reaction.

For this reason, the shape, processability, and other physical properties that can be realized are naturally limited, and the possibility of industrial utilization of the attractive enzyme function has also been limited.

[Purpose of the invention]

The present invention was made in view of the above-mentioned current situation, and it is an object of the present invention to provide a completely different type of production method and a completely new type of modified enzyme and immobilized enzyme in the field of modified enzymes and immobilized enzymes. With the goal.

[Summary of the invention]

The present invention is an invention of a method for producing a modified enzyme, which comprises covalently bonding an azo compound capable of initiating polymerization to the amino group on the surface of the enzyme using a condensation reagent, and then graft-polymerizing a vinyl monomer thereto. .

Further, the present invention is an invention of a modified enzyme obtained by graft polymerizing a vinyl monomer to the enzyme via an azo compound having polymerization initiation ability introduced into the surface amino group of the enzyme.

Furthermore, the present invention uses polystyrene into which a thiol group has been introduced as a carrier, and after activating this with an SH group activator, a reduced enzyme and a vinyl polymer hybrid are added thereto and reacted and immobilized, This is an invention of a special method for producing a modified enzyme, which is characterized in that the modified enzyme is then purified by reduction and separation.

Furthermore, the present invention uses polystyrene into which a thiol group has been introduced as a carrier, and after activating this with an SH group activator, a reduced thiol group-containing protein is added thereto and reacted and immobilized. This is an invention of a special method for producing a thiol group-containing protein, which is characterized in that the protein is then purified by reduction and separation.

That is, as a result of extensive research in order to achieve the above object, the present inventors succeeded in achieving this by covalently bonding an azo compound with polymerization initiating ability to the amino group on the surface of an enzyme, and graft polymerizing various vinyl monomers onto this. We have discovered that a completely new type of IS enzyme, that is, an enzyme/vinyl polymer hybrid, or a completely new type of immobilized enzyme can be obtained, and have completed the present invention.

Enzymes according to the present invention include any enzyme having a free amino group and are not particularly limited, but examples include various lipases, esterases such as cholinesterase and cholesterol esterase, e.g. uricase, glycerol oxidase, Oxidases such as glucose oxidase, cholesterol oxidase, choline oxidase, for example glucose dehydrogenase. Dehydrogenases such as lactate dehydrogenase and α-hydroxybutyrate dehydrogenase, such as papain,
Examples include proteases such as pepsin, trypsin, chymotrypsin, and elastase, amylases, glucosidases, phosphatases, peroxidases, and galactosidases.

Examples of azo compounds having the ability to initiate polymerization that are introduced into surface amino groups (free amino groups) of enzymes include azo compounds having a group capable of covalently bonding to an amino group, such as a carboxyl group. Representative examples of such azo compounds include 4.4'-azodos(4-cyanovaleric acid) (hereinafter abbreviated as ACV), 2,2-azobis(4-carboxy-2-methyl butyronitrile), etc., but are not limited to these, and any azo carboxylic acid that has the ability to initiate polymerization or an azo compound that has the ability to initiate polymerization and has a group that can covalently bond with an amine group. Either one is fine. Dicyclohexylcarbodiimide, 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (hereinafter referred to as 1)
;', abbreviated as wsc. ), Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate), etc., but the present invention is not limited thereto.

The vinyl monomer to be graft-polymerized to the azo compound having polymerization initiating ability introduced into the surface amino group of the enzyme may be any monomer having a vinyl group, and there is no particular restriction. The physical properties of the resulting hybrid or immobilized enzyme are inevitably greatly influenced by the physical properties of the vinyl polymer material to which it is bound, so the appropriate vinyl monomer should be selected depending on the purpose of use. It goes without saying that there is. That is, when selecting a vinyl monomer in consideration of the solubility and affinity of the resulting modified enzyme in a solvent, the following selection methods can be used.

l) When water solubility and hydrophilicity are expected: acrylic acid and its salts, methacrylic acid and its salts, acrylamide, N-methylacrylamide, N-ethylacrylamide, 2-acrylamido-2-methylpropanesulfonic acid and its salts, Vinyl sulfonic acid and its salts, styrene sulfonic acid and its salts, allyl sulfonic acid and its salts, methalylsulfonic acid and its salts, hydroxyalkyl (meth)acrylates, etc. 2) When oil-solubility and lipophilicity are expected Vinyl chloride , vinyl acetate, styrene, acrylonitrile, acrylic esters, methacrylic esters, etc.

3) When amphipathic properties are expected: N-vinylpyrrolidone, relatively long-chain N-alkylacrylamides (propyl, butyl, hexyl, etc.).

4) When a gel-like product is expected, polyfunctional crosslinkable vinyl monomers such as methylene bisacrylamide, diethylene glycol diacrylate, propylene glycol triacrylate, divinylbenzene, triallyl trimellitate, and Allyl isocyanurate or the like is used in combination, or monomers that tend to self-gel, such as hydroxyalkyl (methacrylate) are used.

Furthermore, if the graft-modified product is expected to be formed into a film, bead, fiber, etc. by secondary processing, the shape of the graft may be freely selected according to the processing characteristics of the respective material which are already widely known.

The modified enzyme of the present invention, ie, the enzyme/vinyl polymer hybrid or immobilized enzyme, can be easily synthesized, for example, as described below. That is, first, an azo compound having polymerization initiation ability (for example, ACV) is added to a buffer solution of an appropriate pH (the lower the pH, the higher the introduction rate of the azo compound, so the pH should be selected appropriately depending on the purpose). A binding agent (for example, WSC) is dissolved, and the mixture is left to stand for several hours, usually at 0°C to 10°C, and then an enzyme is added and left to stand for several hours to several tens of hours to react, thereby obtaining an azo compound-introducing enzyme. Next, the obtained azo compound-introducing enzyme was added to the desired vinyl monomer or its appropriate solvent solution, and after nitrogen substitution, light irradiation was carried out, for example, at 200 W.
Polymerization reaction is carried out using a high-pressure mercury lamp (1 to several hours). After the reaction, the product may be precipitated in a suitable solvent (e.g. acetone, methanol, ethanol, etc.) or concentrated.
It is isolated by a conventional method such as freeze-drying, and subjected to further purification steps if necessary.

In the purification step, first, unreacted enzyme is removed by gel filtration, ultrafiltration, etc., and then the homopolymer of the vinyl monomer used is removed by an appropriate method. There are various methods for removing homopolymers, but for example, when the enzyme used is an enzyme having a free SH group, such as lipase, and the obtained modified enzyme is a soluble modified enzyme, the present inventors According to the method discovered below, which uses thiol (SH) group-introduced polystyrene as a carrier, this can be carried out extremely efficiently and effectively, so separation and purification can be carried out by this method.

The method discovered by the present inventors for separating and purifying a modified enzyme and a homopolymer using an S11 group-containing enzyme is roughly as follows. That is, (1) first, polystyrene is dissolved in a small amount of an organic solvent (for example, carbon tetrachloride, chloroform, carbon disulfide, etc.), and this is dropped little by little into chlorosulfonic acid to cause a reaction. The reaction is usually carried out at room temperature, and a reaction time of about 5 to 10 hours is usually sufficient. After the reaction, the reaction solution is added to a large amount of water or dilute acid to form a precipitate, and the precipitate is removed, washed with water, and dried to obtain polystyrene into which 15O□α groups have been introduced. Next, this product, after being pulverized if necessary, is heated and reacted with zinc-hydrochloric acid (or tin-hydrochloric acid, iron-hydrochloric acid, etc.) for 5 to 12 hours. After reaction, unreacted Z
n is separated and removed, washed with water, and dried to obtain SH group-introduced polystyrene (immobilization carrier). (2) The obtained immobilized carrier is completely treated with a suitable reducing agent such as dithiothreitol (hereinafter abbreviated as DTT), 2-mercaptoethanol, thioglycolic acid, thioglycerol, etc. according to a conventional method. Activation is carried out by reducing and then heating reaction with an SH group activator such as 2,2°-dipyridyl disulfide in a solvent such as methanol or ethanol. (3) On the other hand, mixtures of modified enzymes and homopolymers can be prepared using, for example, 2-mercaptoethanol, DTT
, thioglycolic acid, thioglycerol, etc., to reduce the disulfide bonds in the enzyme and convert the modified enzyme into a reduced form.

(4) The mixture of the reduced modified enzyme and the homopolymer obtained in (3) is mixed with a suitable solvent, for example, if the mixture of the modified enzyme and the homopolymer is water-soluble or amphipathic, the pH is such that the enzyme will not be inactivated. The modified enzyme is dissolved in a buffer solution with a pH of 6.0 to 8.0, added to the activated immobilized carrier obtained in step (2), and stirred or shaken at room temperature for 5 to 12 hours to bind the modified enzyme to the carrier via disulfide bonds. to be fixed.

(5) The enzyme does not deactivate the carrier on which the modified enzyme is immobilized.
After thoroughly washing with a buffer solution of pH 6.0 to 8.0 to remove impurities that are not bound to the carrier, that is, homopolymers,
An elution buffer containing a reducing agent such as DTT, 2-mercaptoethanol, thioglycolic acid, or thioglycerol is added to this, and the mixture is stirred or shaken at room temperature for several tens of minutes to reductively cleave disulfide bonds and modify the enzyme. is released from the carrier, and then desalted by gel filtration or the like to obtain the desired purified modified enzyme (enzyme/vinyl polymer hybrid or immobilized enzyme). Also, a book! The blue manufacturing method is SH
It is also extremely effective as a method for purifying proteins in general that have thiol groups (removing proteins that do not have thiol groups and other impurities).

Depending on the purpose of use, the graft reaction product [enzyme/
It goes without saying that the mixture of the vinyl polymer hybrid (or immobilized enzyme) and the vinyl monomer homopolymer used may be used as is without any purification.

Among the modified enzymes of the present invention thus obtained, for example, in the case of oil-soluble, lipophilic or amphiphilic hybrids, organic polyethylene glycol modifying enzymes (hereinafter abbreviated as PEG-modifying enzymes) are used. It can be a solvent-soluble enzyme, has improved thermal stability by hybridization, and has the following characteristics.

1) Since enzymatic reactions can be carried out in an organic solvent system, lipophilic substances can be efficiently treated with enzymes.

2) The equilibrium of the reaction can be shifted.

(For example, by using a hydrolase as a catalyst for a condensation reaction.) 3) Reactions that are impossible in aqueous systems become possible.

(For example, changing stereoselectivity or condensing D-amino acids, etc.) Furthermore, the modification method of the present invention has the following advantages over the PEG modification method.

1) There is a wide range of applications because the choice of vinyl mercury is free and a wide variety of hybrids can be synthesized by changing the type of mercury.

2) Since a low-molecular azo compound is introduced first, the introduction rate is higher and more efficient than when directly introducing a 5-polymer modifier. 3) It is possible to introduce and modify a specific site of an enzyme molecule. .

In addition to its general use as a hybrid enzyme, the modified enzyme according to the present invention can be used in biosensors, bioreactors,
Various uses such as denatured protein materials in general can be expected.

EXAMPLES Below, the present invention will be explained in more detail with reference to examples, but the present invention is not limited in any way by these examples.

(Example) Example 1 Production of ACV-introduced riboprotein lipase ACV 12mg or 24B and wscsrng or 1
6 mg were dissolved in various p++ buffers and incubated at 5°C for 2
After standing for an hour, lipoprotein lipase (hereinafter abbreviated as LPL) lomg was added and the mixture was allowed to stand still for reaction at 5°C for 24 hours. (12+ng of ACV corresponds to 20 times the equivalent of the 7 amine groups contained in LPL,
In the case of GV 24tag, this corresponds to 40 times the equivalent. ) Table 1 shows the LPL when the ACV amount and pH are changed respectively.
ACV introduction rate (determined by quantitative determination of amine groups)
shows.

Table 1 *The amino group was quantified by utilizing the fact that the amino group reacts with 2,4,6-dolinitrobenzenesulfonic acid to form a substance having maximum absorption around 350 mm.

As is clear from Table 1, the introduction rate of AGV in the pH range of 5.4 to 9.4 is over a wide range of 40 to 80%, and the lower the pl+, the higher the introduction rate. .

Example 2. Poly N-vinylpyrrolidone kraft LPL
Production of 0.493 mol/I1. of N-vinylpyrrolidone (hereinafter abbreviated as NVP). AC'h 4-person LPL with an introduction rate of 39.7% obtained in Example 1 was added to the aqueous solution at an initiator concentration of 4,
z 8 μmol/, (l), after nitrogen substitution,
Light was irradiated for 1 hour at room temperature using a 200W high-pressure mercury lamp.

After the reaction, the product was recovered by precipitating it in acetone and was purified using Am1con PM-30 (trade name of Amicon Co., Ltd.).
Unreacted LPL was removed by ultrafiltration using a membrane to obtain poly-N-vinylpyrrolidone (hereinafter abbreviated as PNVP) grafted LPL (including NVP homopolymer). Tatami amount 312n+g Example 3, pN
vpri-y Heptadated LPL Manufactured by Saba (NVP homopolymer #) (1) Synthesis of thiol group-introduced polystyrene (immobilization carrier) Polystyrene log with a degree of polymerization of 2,000 was dissolved in a small amount of carbon tetrachloride. The mixture was gradually added dropwise to 100% of chlorosulfonic acid with stirring, and after the dropwise addition, the mixture was stirred and reacted at room temperature for 6 hours. After the reaction, the reaction solution was gradually poured into 1% 11C1 to form a precipitate, which was collected, washed with water, and dried to obtain 17.8 g of polystyrene into which -502α groups were introduced. 15g of this
was crushed finely, and put into 100 d of concentrated hydrochloric acid together with 10 g of zinc, and reacted with stirring at 70 to 80° C. for 8 hours. After the reaction,
Separate from unreacted zinc by decantation, remove suspended matter, wash with water, and dry to make thiol group-introduced polystyrene 13.
．． 6g was obtained. As a result of elemental analysis, the SH group introduction rate (with respect to the benzene ring) was about 30%.

(2) Activation of the immobilization carrier 5 g of the thiol group-introduced polystyrene powder obtained in (1) was
TT 50mM, O, IM borate buffer (pH 7,8
＞20m1! was added and shaken for 15 minutes to complete the reduction reaction. The product was collected and thoroughly washed with water to remove DTT, and then 10% of 2,2-dipyridyl disulfide was added to the product.
40ml of 0mM ethanol solution was added, and the mixture was immersed at 80°C for 3 hours and left to cool overnight in a water bath. The product was collected, washed with ethanol, and then washed with 0.1M hologram. 2 buffer (pH 7
, 8) suspended in the middle.

(3) Reduction of protein disulfide bonds PNVP-grafted mixture of LPL and NVP homopolymer 72.3
2-mercaptoethanol was dissolved in 5 ml of 250 mM 2-mercaptoethanol, stirred and reacted at room temperature for 12 hours, and the 2-mercaptoethanol was removed by freeze-drying.
Blend of P-grafted LPL and NVP homopolymer 7
1.0 g was obtained.

(41PNVP grafted 1. PL < 7) 71.0 g of the mixture of the close-type PNVP grafted LPL obtained in purification (3) and the NVP homopolymer was dissolved in a boric acid buffer solution of pH 7.8, and (2) It was added to the suspension of the obtained activated immobilization carrier and shaken at room temperature for 8 hours to immobilize only the protein component on the carrier via disulfide bonds. Isolate the carrier on which the protein component is immobilized, and add 15 ml of boric acid buffer at pH 7.8 to it! The homopolymer was washed and removed by repeating the operation of adding and shaking for 10 minutes at room temperature three times.

This was then added with DTT in 20mM borate buffer (pH 7).
, 8) was added and shaken at room temperature for 30 minutes to reduce and separate the protein component from the carrier. The carrier was removed by centrifugation, and desalted by gel filtration using 5ephadex G-10 (trade name of Pharmacia).
PNVP-grafted LPL 14.4IIg was obtained. The degree of polymerization of the obtained PNVP-grafted LPI was 700,
Protein content was 6.61%. A part of the IR spectrum (1400-1900 cm-') is shown in FIG.

In addition, ACv introduction rate 39.7% (7) ACV introduction LPL
(1) Instead, PNVP-grafted LPL was produced in the same manner as in Example 2 using ACV-introduced LPL with an introduction rate of 41.4%, and purified in the same manner as in Example 3. PNVP-grafted LPL with an actual protein content of 7.80% was obtained.

Reference Example 1, Production of PEG-modified Enzyme [Y, Inada, et: al.
Biochem.

Bjophys, Res, Commun,, 12
2.845-850 (1984)].

2.4-bis(0-methoxypolyethylene glycol)
-6-chloro-s-triazine (hereinafter, activated PEG2
It is abbreviated as. ) [Made by Wako Pure Chemical Industries ■] 0.8g and 1,
PL 20mg was added to 0.4M borate buffer (pH 1O, 0
After stirring and reacting at 37°C for 1 hour, add 100ml of 0.1M borate buffer (pH 8,0).
1 was added to stop the reaction. Note that the amount of activated PEG2 used is equivalent to 20 times the amount of LPL. After reaction, Am1
Using con PM-30 (trade name of Amicon Inc.) nq,
Unreacted activated PE02 and LPL were removed by ultrafiltration and lyophilized to obtain 11.4 mg of PEG-modified LPL. The PEG introduction rate was 50% of the total amino groups in LPL.

Reference Example 2゜PNVP-modified LPL obtained in Example 3 (introduction rate 39.7
% and that with an introduction rate of 41.4%) and the solubility of the PEG-modified LPL obtained in Reference Example 1 in chloroform were determined. The results are shown in Table 2 along with those of unmodified LPL.

Table 2 *Modified LPL or unmodified LPL I button g was added to chloroform 1], fii, the precipitate was separated by centrifugation after shaking, and weighed after drying, and the decrease was taken as the solubility (mg/).

In addition, taking advantage of the fact that LPL can catalyze the condensation reaction, which is the reverse reaction of the hydrolysis reaction, in an organic solvent, we used n-amyl alcohol and n-caprylic acid as substrates, and in chloroform. Table 3 shows the results of measuring the activities of PNVP-modified LPL and PEG-modified LPL and comparing them with that of unmodified LPL.

Table 3 **0.92M n-amyl alcohol and 0.92M n-caprylic acid in chloroform were reacted at 37°C.
The amount of alcohol reduction was measured by tracking with gas chromatography.

As is clear from Table 3, the activity of unmodified lipase in chloroform was extremely low and unsuitable for practical use, but PEG modification increased the activity more than 100 times, and the activity of PNVP modified according to the present invention was 1, P], an even greater activity was observed.

Example 4. Production of polystyrene-grafted LPL 5 ml (4,545 g) of styrene was prepared in the same manner as in Example 1. A CV introduction rate of about 40% <7) A CV d
Add human LPL to an initiator concentration of 37.3 nmol/IL, and use a 200W high-pressure mercury lamp at 30℃ for 2 hours.
A polymer was obtained by irradiation with light for a period of time. The reaction rate of ACV-introduced LPL was 13.6%. Styrene reaction rate 0.23%. The IR spectrum of the obtained polymer is shown in FIG.

Reference Example 3 Enzyme activity in chloroform was determined in the same manner as in Reference Example 2, using the polystyrene-modified LPL obtained in Example 4 without making M. The results are shown in Table 4 along with those of unmodified LPL.

Table 4 As is clear from Table 4, even when polystyrene was grafted, the activity increased approximately 60 times compared to unmodified LPL, and even when modified with a hydrophobic polymer, the activity in organic solvents was significantly increased. I knew it was going to rise.

〔Effect of the invention〕

As described above, the present invention provides a completely different type of production method from conventional methods in the field of modified enzymes and immobilized enzymes, and a completely new type of modified enzyme and immobilized enzyme obtained thereby. For example, it has remarkable effects in the following points.

1) By using the modified enzyme of the present invention, lipophilic substances can be efficiently treated with enzymes, and reactions that are impossible in aqueous systems can be performed. 2) By using the modified enzyme of the present invention, , the equilibrium of the reaction can be shifted, and for example, a hydrolase can be used as a catalyst for the condensation reaction. 3) According to the production method of the present invention, the vinyl monomer to be introduced can be freely selected, so thousands of types can be used. It is possible to synthesize various hybrids by changing the polymer modifier, and the introduction rate of the modifier is higher and more efficient than the conventional method of directly introducing a polymer modifier.

[Brief explanation of the drawing]

Figure 1 shows I of the modified enzyme according to the present invention obtained in Example 3.
A part of the R spectrum (1400 to 1900 cm-') is shown. Moreover, FIG. 2 shows the IR spectrum (full spectrum) of the modified enzyme according to the present invention obtained in Example 4. Patent applicant: Aiko Pure Chemical Industries, Ltd. Figure 1 Procedural Amendment April 6, 1999

Claims (4)

[Claims] (1) A method for producing a modified enzyme, which comprises covalently bonding an azo compound capable of initiating polymerization to an amino group on the surface of the enzyme using a condensation reagent, and then graft-polymerizing a vinyl monomer thereto. (2) A modified enzyme obtained by graft-polymerizing a vinyl monomer onto the enzyme via an azo compound having polymerization initiation ability introduced into the surface amino group of the enzyme. (3) Polystyrene into which a thiol group has been introduced is used as a carrier, and after activating this with an SH group activator, a reduced enzyme/vinyl polymer hybrid is added to this, reacted and immobilized, and then this A method for purifying a modified enzyme, the method comprising purifying a modified enzyme by reducing and separating the enzyme. (4) Use polystyrene into which thiol groups have been introduced as a carrier, activate it with an SH group activator, add a reduced thiol group-containing protein to it, react and immobilize it, and then A method for purifying a thiol group-containing protein, the method comprising purifying it by reduction and separation.