JPS6217517B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an immobilized enzyme and a method for producing the same, and more specifically to the following general formula () (Here, R ¹ is a hydrogen atom or a lower alkyl group, B is

[expression] or

[Formula] R ² , R ³ , and R ⁴ are lower alkyl groups (which may contain substituents), X is a negative group that forms a salt with ammonium nitrogen, and A is a group that connects B and the nitrogen atom of the amide group. , respectively. ) This invention relates to an immobilized enzyme obtained by adsorbing an enzyme to modified polyvinyl alcohol (hereinafter referred to as PVA) containing a copolymerized unit represented by the following formula, and a method for producing the same. Conventionally, when producing various substances by fermentation using catalytic reactions by enzymes, so-called "immobilized enzyme technology" has been used to immobilize enzymes to facilitate their separation from reaction products and enable continuous operation. It has been known. Immobilization methods include a cross-linking method in which enzymes are bonded to each other using a cross-linking agent such as glutaraldehyde, a carrier bonding method in which the enzyme is bonded to an ion exchange resin or glass beads, or a method in which enzymes are bonded to collagen membranes or gel-like synthetic polymers. Another method is to enclose the enzyme, and the enzyme immobilized by these methods is usually in the form of particles, membranes, or gels, and is used by filling it into a reaction tank such as a column. On the other hand, in order to make the immobilized enzyme even easier to handle, methods of making it into a fiber shape are also being considered.
In this issue, a technology for immobilizing enzymes on aminoacetalized PVA fibers is disclosed. However, the method of converting PVA into aminoacetal requires expensive raw materials, the process is complicated, and it is difficult to implement industrially.
A concentrated aqueous solution of PVA tends to cause an insolubilization reaction depending on the conditions, and in that case, the aqueous solution becomes unstable and non-uniform, making spinning difficult. The present inventors have developed PVA, which has excellent adsorption and fixation performance for enzymes and can form fibers industrially at low cost and stably.
As a result of our study to develop a system polymer, we found that vinyl acetate and a polymerizable monomer represented by the following general formula (a) (Here, R ¹ is a hydrogen atom or a lower alkyl group, B is

[expression] or

[Formula] R ² , R ³ , R ⁴ are lower alkyl groups (which may contain substituents), X is a negative group that forms a salt with ammonium nitrogen, A is the nitrogen atom in B and the nitrogen in the amide group Each refers to a group that connects atoms. ) Modified PVA obtained by partially or completely saponifying the vinyl acetate units in a copolymer with PVA is easy to produce industrially, and its aqueous solution has excellent stability. The enzyme can be easily immobilized on the fiber by immersing the obtained fiber in an enzyme aqueous solution, and this fibrous immobilized enzyme is convenient to handle and exhibits high enzyme activity, making it suitable for the fermentation industry. The present invention was completed after confirming that it is extremely useful. The details of the method for producing modified PVA containing copolymerized units represented by the above general formula () are as shown in the specification of Japanese Patent Application No. 54-89078. It is most effectively and simply produced by copolymerizing the polymer with vinyl acetate and then saponifying the vinyl acetate unit. In the formula (a), R ¹ is a hydrogen atom or a lower alkyl group, but usually a hydrogen atom or a methyl group is preferable, and a hydrogen atom is more preferable because it has a high polymerization rate in the copolymerization reaction with vinyl acetate. preferable. B is a tertiary amino group

[Formula] or quaternary ammonium salt

[Formula] where R ² , R ³ and R ⁴ represent lower alkyl groups which may contain substituents, and X represents a negative group that forms a salt with ammonium nitrogen.
R ² , R ³ , and R ⁴ are preferably all methyl groups for normal purposes, but for special purposes they may be lower alkyl groups such as ethyl or propyl groups, or for the purpose of imparting reactivity, methylol groups or cationic groups. Lower alkyl groups containing substituents such as aminoalkyl groups are also used for the purpose of improving density. As X, a halogen atom such as chlorine, sulfur, or iodine, or CH ₃ OSO ₃ or CH ₃ C ₆ H ₄ SO ₃ is preferable, and a chlorine atom is particularly preferable from the economical, safety, and property standpoints of modified PVA. It is preferable for the amino group to be in the form of a quaternary ammonium salt in the production of modified PVA, but it can also be produced in the form of a tertiary amine and the effects of the present invention can be achieved. As the group A that connects the nitrogen atom in the amino group B and the nitrogen atom of the amide group, any group containing a stable bond can be used, but a linear or branched aliphatic group is usually used. . Among the aliphatic groups, monomers in which A is an aliphatic group with a side chain as shown in the formula in parentheses in the following formula have particularly high amide bond stability, and their synthesis is economical. It is particularly advantageous for two reasons. Here, R ⁶ and R ⁷ are alkyl groups having 10 or less carbon atoms, R ⁸ is a hydrogen atom or an alkyl group having 10 or less carbon atoms, and n is an integer from 0 to 2. Among the monomers represented by the above general formula (A), the following are specific examples of monomers in the form of tertiary amines. N-(2-dimethylaminoethyl)acrylamide, N-(2-dimethylaminoethyl)methacrylamide, N-(3-dimethylaminopropyl)acrylamide, N-(3-diethylaminopropyl)acrylamide, N-(3-dimethyl aminopropyl) methacrylamide, N-(3-
diethylaminopropyl) methacrylamide, N
-(1,1-dimethyl-3-dimethylaminopropyl)acrylamide, N-(1,1-dimethyl-3-dimethylaminopropyl)methacrylamide, N-(1,1-diethyl-3-dimethylaminobutyl)acrylamide ,N-(1-methyl-
1,3-diphenyl-3-diethylaminopropyl)methacrylamide, N-(3-dimethylaminohexyl)acrylamide, N-(3-methylethylaminopropyl)methacrylamide. Among the monomers represented by the above general formula (a), those in the form of quaternary ammonium can be easily obtained by quaternizing the above-mentioned tertiary amine type monomer with the following quaternizing agent. be able to. Dialkyl sulfate, e.g. dimethyl sulfate, diethyl sulfate, dipropyl sulfate, alkyl or arylsulfonic acid C ₁ ~
_C4 esters, e.g. methyl-, ethyl-, propyl- or butyl esters such as methanesulfonic acid, benzenesulfonic acid or toluenesulfonic acid, benzyl halides e.g. benzyl chloride or benzyl bromide, alkyl halides e.g. methyl chloride, bromide Examples include methyl, methyl iodide, ethyl chloride, ethyl bromide, or ethyl iodide. The following four types of monomers among the examples mentioned above as monomers represented by the above general formula (A)

[Table] is superior in producing the modified PVA used in the immobilized enzyme of the present invention from the viewpoints of polymerization rate, stability of the amide group, and economical efficiency during monomer production. Modified PVA includes both completely saponified vinyl acetate units and partially saponified vinyl acetate units. The amount of modified groups in the modified PVA used in the immobilized enzyme of the present invention, the degree of saponification, or the degree of polymerization of the modified PVA are appropriately selected depending on the purpose and are not particularly limited. It is important to skillfully combine these three elements. For general purposes, the amount of copolymerized units containing modifying groups is preferably in the range of 0.5 to 15 mol%, the degree of saponification is in the range of 80 to 100 mol%, and the degree of polymerization is in the range of 500 to 3000. The modified PVA thus obtained is fibrous,
It is possible to form the immobilized enzyme of the present invention by shaping it into a sponge-like, gel-like, etc. shape and then reacting it with an enzyme, but among these, the immobilized enzyme in the form of a fiber is particularly superior in its ease of handling. Since it is particularly important in the present invention, the immobilized enzyme using fibrous modified PVA will be explained below. Modified PVA is heated and dissolved in water to make a 10-50% aqueous solution, and the stock solution is prepared at a temperature of 60°C to 160°C under normal pressure or high pressure, and then processed using the usual dry spinning method or wet spinning method. Spun by spinning. When preparing this stock solution, it is also possible to mix and spin unmodified ordinary PVA in order to control the amount of modifying groups. The spinning tube in the dry spinning method is
Heated air at 90° to 250°C is used, and an example of a spinning bath in the wet spinning method is 200 to 450 g/
An aqueous solution of sulfur salt at a concentration of 30-50°C is used. The spun modified PVA is then heated at 150°~260°
After stretching 4 to 12 times in heated air at 200 to 260°C. The fibers thus obtained can be used as they are, or may be formalized or acetalized to improve water resistance or adjust the degree of swelling. It can also be used in the form of composite fibers containing the copolymer as one component. The thickness of the fibers can be arbitrary depending on the purpose, but is usually preferably about 0.5 to 500 deniers. The spinning method, draw ratio, heat treatment, formalization, and fiber thickness must be selected appropriately depending on the composition of the modified PVA raw material, and are adjusted to provide an appropriate degree of swelling and water resistance. In order to adsorb enzymes to the fibrous modified PVA obtained in this way, the fibers are cut as appropriate depending on the purpose, or after being woven or knitted into a cloth shape, the fibers are adjusted to an appropriate pH depending on the properties of the enzyme using a buffer solution. The enzyme is adsorbed and immobilized on the fibrous modified PVA by immersing it in an aqueous enzyme solution adjusted to the desired temperature and allowing it to stand for a required period of time or being shaken or stirred to form an immobilized enzyme. There are no particular restrictions on the enzyme used in the immobilized enzyme of the present invention, and many enzymes can be used depending on the purpose. Examples include inverdase, glucoamylase, glucose isomerase, glucose oxidase, urease, ribonuclease, deoxyribonuclease, protease, lactate dehydrogenase, L-amino acid oxidase, steroid esterase, pyruvate kinase, acetylcholinesterase, alkaline phosphatase, lactase, leucine amino acid. These include, but are not limited to, peptidase, aminopeptidase, trypsin, papain, fuicin, pronase, aminoacylase, and asparaginase. Further, prior to adsorption, it is optional to appropriately modify the enzyme, such as succinylation, for the purpose of improving the adsorption and immobilization ability of the enzyme. The function of the obtained immobilized enzyme is similar to that of the original enzyme, but since it is solidified, it has the advantage of being easy to handle, separate from the reaction solution, and reuse. The activity of the immobilized enzyme usually has an expression rate of 40% or more compared to before immobilization. The present invention will be explained below with reference to Examples, but these Examples are not intended to limit the present invention in any way. Example 1 Trimethyl-3-(1-acrylamide-1,
Saponifying a copolymer of 1-dimethylpropyl) ammonium chloride and vinyl acetate to obtain 5 mol% of trimethyl-3-(1-acrylamido-1,1-dimethylpropyl) ammonium chloride units.
The degree of saponification of vinyl acetate units is 99.9 mol%, and the viscosity of a 4% aqueous solution at 20°C (as measured by a Bruckfield viscometer; the same applies hereinafter) is 30CP.
(centipoise) modified PVA was synthesized. A 35% aqueous solution of this modified PVA is used as a stock solution, which is heated to 130°C and extruded from a metal plate into heated air at 150°C for dry spinning.The resulting fibers are stretched 7 times at 230°C and then A 2-denier fibrous modified PVA was obtained by heat treatment at 240°C. Inverdase aqueous solution adjusted to pH 6 with phosphate buffer to 1g of this fiber
100 g (containing 10,000 international standard units) was added, and the enzyme was fixed by shaking at 30°C for 24 hours.
Next, after thoroughly washing the fibers with water, the immobilization rate of invertase was measured and found to be 89%. When 100 g of a 2% sucrose aqueous solution was added to the thus obtained fibrous immobilized enzyme, the enzyme activity expression rate was determined from the conversion rate to glucose at 30°C.
The enzyme activity was 76% when the activity of the raw enzyme was taken as 100%. Example 2 A copolymer of N-(1,1-dimethyl-3-dimethylaminopropyl)acrylamide and vinyl acetate was saponified to produce N-(1,1-dimethyl-3-
Contains 6 mol% of acrylamide units (dimethylaminopropyl), and the degree of saponification of vinyl acetate units is
The viscosity of a 99.7 mol%, 4% aqueous solution at 20℃ is
A modified PVA of 20CP was synthesized. 40 of this modified PVA
% aqueous solution as a stock solution, a fibrous modified PVA of 3 denier was obtained by a dry spinning method according to Example 1.
1 g of this fiber was immersed in 0.5N hydrochloric acid overnight and then thoroughly washed with water. 50 g of glucose isomerase aqueous solution adjusted to pH 8 with phosphate buffer per 1 g of this fiber
(containing equivalent to 10,000 units) was added, and the enzyme was fixed by shaking at 30°C for 24 hours. Next, after thoroughly washing the fibers with water, the enzyme immobilization rate was measured and found to be 91%.
It was hot. 100 g of a 2% glucose aqueous solution was added to the fibrous immobilized enzyme thus obtained, and the enzyme activity expression rate was determined from the rate of isomerization to fructose at 30°C, and was found to be 68%. Example 3 A copolymer of trimethyl-3-(1-methacrylamido-propyl) ammonium chloride and vinyl acetate was saponified to obtain a copolymer containing 3.5 mol% of trimethyl-3-(1-methacrylamido-propyl) ammonium chloride units. , a modified PVA with a degree of saponification of vinyl acetate units of 98.5 mol% and a 4% aqueous solution having a viscosity of 25CP at 20°C was synthesized. This degeneration
Using a 38% aqueous solution of PVA as a stock solution, 2-denier fibers were modified by dry spinning according to Example 1.
Got PVA. Add 1g of this fiber to phosphate buffer
50 g of an aqueous urease solution (containing 10,000 units) adjusted to pH 8 was added, and the mixture was shaken at 30° C. for 24 hours to carry out enzyme fixation. The immobilization rate of urease was 78%. 100 g of a 2% aqueous urea solution was added to the fibrous immobilized enzyme thus obtained, and the enzyme activity expression rate was determined from the rate of ammonia generation at 30°C, and was found to be 90%. Example 4 A copolymer of N-(3-dimethylaminopropyl)methacrylamide and vinyl acetate was saponified to contain 3 mol% of N-(3-dimethylaminopropyl)methacrylamide units, and a copolymer of vinyl acetate units was saponified. A modified PVA with a degree of oxidation of 97.0 mol% and a 4% aqueous solution having a viscosity of 30CP at 20°C was synthesized. This degeneration
A wet spinning method is performed in which a 16% aqueous solution of PVA is used as a stock solution, heated to 94°C, and extruded from a metal plate into a 35°C bow salt solution.The resulting fiber is stretched 8.5 times at 200°C, and then A 2-denier fibrous modified PVA was obtained by heat treatment at ℃. Inverdase aqueous solution adjusted to pH 6 with phosphate buffer to 1g of this fiber
100g (containing 10,000 units equivalent) was added and shaken at 30°C for 24 hours to carry out enzyme fixation. The immobilization rate of inverdase was 92%. 100 g of a 2% sucrose aqueous solution was added to the fibrous immobilized enzyme thus obtained, and the enzyme activity expression rate was determined from the conversion rate to glucose at 30°C, which showed an activity of 75%.

Claims (1)

[Scope of Claims] 1. An immobilized enzyme obtained by adsorbing an enzyme to modified polyvinyl alcohol containing a copolymerized unit represented by the following general formula (). (Here, R ¹ is a hydrogen atom or a lower alkyl group, B is [Formula] or [Formula] R ² , R ³ , R ⁴ are lower alkyl groups (which may contain substituents), X is ammonium nitrogen and salt (A means a group that connects B and the nitrogen atom of the amide group, respectively.) 2. The fixation according to claim 1, in which the modified polyvinyl alcohol is in the form of fibers. enzyme. 3. Modified polyvinyl alcohol obtained by partially or completely saponifying vinyl acetate units in a copolymer of vinyl acetate and a polymerizable monomer represented by the following general formula (A) is spun to make fibers. A method for producing an immobilized enzyme, which comprises forming an immobilized enzyme, and immersing the immobilized enzyme in an enzyme aqueous solution to adsorb the enzyme onto fibrous modified polyvinyl alcohol. (Here, R ¹ is a hydrogen atom or a lower alkyl group, B is [Formula] or [Formula] R ² , R ³ , R ⁴ are lower alkyl groups (which may contain substituents), X is ammonium nitrogen and salt (A means a group that connects the nitrogen atom in B and the nitrogen atom in the amide group, respectively.)