WO2005073370A1

WO2005073370A1 - Enzyme composite

Info

Publication number: WO2005073370A1
Application number: PCT/JP2005/000446
Authority: WO
Inventors: Yukio Nagasaki; Akiko Watanabe; Michihiro Iijima; Kazunori Kataoka; Kenji Yoshinaga
Original assignee: Transparent Inc.
Priority date: 2004-01-31
Filing date: 2005-01-11
Publication date: 2005-08-11
Also published as: JPWO2005073370A1

Abstract

A composite of an enzyme with a specific polymer. It optionally has fine particles coexistent therewith. The polymer especially suitably is poly(ethylene glycol)-block-poly(aminoethylmethacrylic acid), which can significantly heighten the thermal stability of the enzyme.

Description

Specification

Enzyme complex technology

The present invention relates to a complex of an enzyme and a water-soluble high molecule, which may optionally contain microparticles.

Background art

Enzymes that cause extremely selective chemical reactions under mild conditions have been widely used industrially as catalysts for selective synthesis reactions and the like. However, the enzyme, which is a protein, was easily deactivated by a temperature, pH, or the like exceeding a certain range, and it was extremely difficult to separate it from the product after the reaction. Thus, the concept of immobilized enzymes was discovered, in which enzymes are immobilized on a water-insoluble carrier and modified so that they can be reused. Some enzymes have been industrialized (for example, “immobilized enzymes”). , Ichiro Chibatake, Kodansha).

However, when the enzyme is immobilized on a water-insoluble carrier or substrate, it is generally unavoidable that the enzyme activity is significantly reduced, and it may be possible to impart a certain degree of heat resistance to the enzyme. It does not achieve excellent heat resistance.

In recent years, colloidal gold (colloidal gould) particles have been used as carriers, suggesting that the enzyme can be stabilized while maintaining its original state. Gates have been proposed (A. Gole et al, Langmuir 2001, Vol. 17, 1674-1679). In addition, a bio-copper in which pepsin is immobilized on a carrier in which gold nanoparticles are assembled on zeolite functionalized with amine Njugeto, together exhibit excellent enzyme activity has also been reported to exhibit good stability at high _P H and temperature (K. Mukhopadhyay et al, Langmuir 2003 , Vol.19, 3858-3863). Furthermore, when an enzyme, such as endoglucanase, is immobilized on a polyurethane-microsphere core-shell structure of gold nanoparticles, the immobilized enzyme retains its activity and is free in aqueous solution. It has been reported to show improved thermostability compared to enzymes (S. Phadtare et al, Biotechnol. Prog. 2004, Vol. 20, 1840-1846). It has been suggested that these enzymes immobilized on the gold surface exist in the enzyme reaction system in almost a free state in almost half.

An enzyme complex or conjugate developed to stabilize the enzyme by binding a water-soluble polymer to the enzyme without using a water-insoluble carrier has also been proposed. . Representative examples include the following. It has been reported that treatment of ct-amylase with polyamine, spermidine or cadaverine can enhance enzyme activity and increase stability (RD Due et al, J. Molecular Catalysis). , 1980, Vol. 7, 443-456). It is known that treatment of lipase with polyamine and dimethyl suberimidate generally improves the enzyme activity and, for example, increases the heat resistance at 50 ° C (M. Kawase et al, Agrio. Bio. Chem. 1990, Vol. 54, 2605-2609). It has been reported that the non-covalent binding of poly (ethylene oxide) to an enzyme under high pressure reduces the activity considerably as compared to the native enzyme, but can slightly increase the thermal stability. IN Topohieva et al, Bioconjugate Chem., 2000, Vol.11, 22-29). Also, some polyamines (eg, bis (ethylamino) octane, 1,8-octanediamine, 1,5-bis (ethylamino) pentane, 1,8-bis (ethylamino) 14-azaoctane, spermidine It is also known that modification of the enzyme T7 RNA polymerase with Agmethine, etc.) maintains the enzyme activity but decreases the activity in the order described (M. Iwata et al, Bioorganic and Medical Chemistry, 2000, Vol. 8, 2185-2194).

In addition, it is known that the enzyme's Km value can be increased by chemically covalently binding the enzyme to poly (ethylene oxide) or poly (ethylene oxide) -block-poly (propylene oxide) (IN Topohieva, Polyethyrene glycol, 1997, 193-206).

Disclosure of the invention

As described above, non-covalent binding of an enzyme to a polyamine or colloidal gold particle or a carrier carrying colloidal gold can stabilize the enzyme while maintaining a certain level of enzyme activity. Achieved. However, there remains a need for further stabilization, especially for obtaining enzyme conjugates or conjugates with improved thermostability while retaining the original enzyme activity. Improvement of thermal stability is important from the viewpoint that the reaction rate can be increased when performing industrial enzymatic conversion of a substrate compound.

Thus, the present inventors have conducted intensive studies, and found that a certain type of water-soluble polymer, specifically, in some cases in the presence of microparticles, a charged group or hydrophobic group is added to the end of one hydrophilic polymer chain. A polymer having one or many groups, It has been found that an enzyme complex or conjugate which can meet the above-mentioned needs can be obtained by non-covalently treating an enzyme with the enzyme.

Accordingly, the present invention provides an enzyme complex in which a polymer having one or many chargeable groups or hydrophobic groups at the ends of a hydrophilic polymer chain and an enzyme are non-covalently bound.

In another aspect of the present invention, a polymer and an enzyme having one or many charged groups or hydrophobic groups at the ends of a hydrophilic polymer chain are non-covalently bound to the surface of water-insoluble microparticles. Provide an enzyme complex.

According to the prior art, for example, I. N. Topoh ieva et al, Bioconjugate Chem., 2000, Vol. 11, 22-29), polyamines having a large molecular weight such as spermidine and agmethine are known. Although the degree of the activity decrease is larger than that of the enzyme treated with low-molecular bis- or diamine, according to the present invention, a polyamine having a poly (ethylene dalicol) chain having a considerably large molecular weight was used. Even in such a case, the enzyme activity of the obtained enzyme complex is substantially the same as that of the original enzyme, and particularly, the heat stability is significantly increased.

Hereinafter, the present invention will be described in more detail.

The enzyme complex according to the present invention is formed non-covalently from an enzyme and a certain polymer in the presence of microparticles in some cases, and at least depending on the treatment such as stirring or centrifugation in an aqueous medium. A structure in which components are retained without separation, including what is commonly referred to as a complex, conjugate, or bioconjugate. Any enzyme constituting the complex may be used as long as it meets the purpose of the present invention. Enzymes may be used, but preferred ones that are currently used industrially are preferred. Examples of such enzymes include, but are not limited to, proteases such as pepsin, trypsin, chymotribsine, collagenase, keratinase, elastase, subtilisin, papain, aminopeptidase, carboxypeptidase, etc. , Gastric lipase, puncture lipase, plant lipase, phospholipase, etc. Can be mentioned.

Polymers that can be used in the present invention that bind non-covalently to such enzymes or microparticles include aqueous media (pH buffered aqueous solutions under physiological or non-physiological conditions, water-miscible An organic solvent, for example, an aqueous solution containing a mixture of methanol, ethanol, acetone, dimethinolehonoremamide, dimethyl sulfoxide, and acetonitrile, etc.). It is a polymer having one or many charged groups or hydrophobic groups at its terminals. Specifically, the hydrophilic polymer chains are composed of poly (ethylene glycol), poly (2-hydroxyhexyl methacrylate), poly (2-hydroxyacrylic acid), poly (vinyl alcohol), Poly (acrylamide), poly (methacrylamide), poly (N, N-dimethylaminoacrylamide), poly (N, N-dimethylmethacrylamide), poly (vinylpyrrolidone), Selected from the group consisting of agarose and dextran, and wherein the chargeable group is derived from a low-molecular-weight amine compound containing one or two primary, secondary or tertiary amines in the molecule, or primary or secondary. Alternatively, it is derived from a polymer chain containing one or two tertiary amines in the side chain or main chain of the repeating unit, or has a hydrophobic group of poly (lactic acid), poly (glycolic acid), or poly (petitic acid). ) Is a polymer derived from polyester. Particularly preferred polymers are those in which the hydrophilic polymer chains are derived from poly (ethylene glycol) or poly (ethylene oxide). Specific examples include a polymer represented by the following formula (I).

R ³ -L! - in X (I) above formula, R ^a is hydrogen, a methyl group, aldehyde group, amino group, carboxyl group, maleimidyl de _{group, p - - (CH 2 CH} 2 O) n - L 2 toluenesulfonyl Group or vinylsulfonyl group,

And L ₂ independently represent a valence bond or a linker; n is an integer from 2 to 20;

X is, N- or N, N- mono- or di-one C i-C ₃ alkyl substituted amino C i-C! ₂ alkyl, to repeat units of 2 to 2 0 0 0 0 oligo or poly (N- or N, N-mono- or di one C -! C ₃ alkyl-substituted aminoethyl meth Ata Li rate) chain, the main chain For example, it is a group derived from a polyamine having a secondary or tertiary amine group, a group selected from the group consisting of a polyethyleneimine chain, a sperdimine chain and a cadaverine chain, or an oligo or polymer chain segment.

Further, more specific polymers include those represented by the following formula.

_{_{_{_{CH 3 OCH 2 CH 2 CH 2}}}} 0- (CH 2 CH 2 0) -

In the above formula, m and n are independently

R is a C 1 -C ₃ alkyl group.

-(CH ₂ C (CH ₃ )) _m -... ((CHg CR ¹ ))-COOCH. CH ₂ N (R ² ) ₂

COOCH ₂ CH ₂ 0— (CH ₂ CH O)-R '

In the above formula, m, n and x are each independently an integer of 2 to 20, 000, R ¹ is hydrogen or a methyl group, R ² is a C_C ₃ alkyl group R ³ is hydrogen or a C—C ₅ alkyl group.

The present invention relates, C i - the term C i ₂ alkyl means 1 to 1 2 straight-chain or branched alkyl carbon atoms.

Hereinafter, more specific examples of the polymers included in the above-mentioned polymers include polyethylene glycol (PEG) -block-poly (2-N, N-dimethylaminoethyl methacrylate) (hereinafter, PEG- PMAMA), PEG-block-poly (2-N, N-methylethylamino methacrylate) (hereinafter abbreviated as PEG-PEAMA), PEG-block-poly (isopropylacrylamide) ) (hereinafter, PEG-abbreviated as PNIPAM), PEG-polyacrylamide Riruami de, PMAMA-PNIPAM, PEAMA-PNIPAM , PVA-PMAMA, PVA - PEAMA Hitoshiryoku S ₀ encompassed

The molecular weight of these polymers is not theoretically limited, but is preferably from 500 to 1,000,000, more preferably from 2,000 to 500,000, and from the viewpoint of ease of synthesis, from 300,000 to 100,000. Force is particularly preferred. These polymers are It is known per se or can be easily obtained by a method according to a known one. For example, PEG-PMAMA, PEG-PEAMA, etc. are described in Y. Nagasaki et al., Macromol. Chem. Rapid Commun. 1997, 18, 927, when introducing various functional groups into the _α -terminal of polyethylene glycol chain. W096 / 32434 (corresponding to USP 5,973,069), W096 / 33233 (corresponding to USP 6,090,317) when adjusting the molecular weight or obtaining a polymer having a hydrophobic polymer chain. 097/06202 (corresponding to USP 5,929,177).

In addition, the microparticles which can be used as a component of the enzyme complex of the present invention are characterized in that the polymer and the enzyme as described above are non-covalently bonded (except for covalent bonds such as electrostatic bond, hydrophobic bond, hydrogen bond, etc.). (Including any bonding mode), and can be used irrespective of the material, origin, etc. as long as the object of the present invention is met. Also, the term “micro” means that the size is sub or several nm to several mm, preferably, 111 to 100, and more preferably, 5 nm to 100 nm. Intended for something. In the prior art, an enzyme bound or immobilized on colloidal gold has a certain degree of dispersion stability in an aqueous medium, but according to the present invention, particles having a size exceeding the colloidal shape are also used as enzymes. In addition, due to the binding of the polymer, extremely good dispersion stability in an aqueous medium can be exhibited.

Representative examples of these microparticles include, but are not limited to, colloidal gold, colloidal silver, colloidal aluminum oxide, colloidal silica, colloidal titanium oxide, and fine magnetic particles. Examples include steel particles and colloidal semiconductors. All such particles are commercially available. It can be used as is or after purification. In addition, semiconductor microparticles include, for example, chlorides (eg, chlorides) of elements of group IIB or IIIB of the periodic table and salts of metal salts of the same group VIB (eg, sodium sulfide). May be reacted in an aqueous medium. Representative examples of the semiconductor fine particles that can be adjusted in this way include 0.30 micro particles (13.6 fine particles).

These enzyme complexes can be prepared by simply mixing and stirring the enzyme and the polymer in the above-mentioned aqueous medium at room temperature, if necessary, for example, under cooling (a few ° C). Since it can be easily prepared in this way, it is one of the advantages of the present invention that the mixed solution can be concentrated or diluted as needed and supplied to a target enzyme reaction system.

In this case, the mixing ratio varies depending on the type of the enzyme and the polymer to be used, particularly the type of the polymer. Therefore, it is usually better to determine the mixing ratio by performing a small experiment. 0001: 1000 to 100: 1 can be used, and 1: 1000 to 10: 1 is more preferable. Further, for example, when PEG-PMAMA or the like having a tertiary amine in the side chain is used, the tertiary amine group is at least 1, preferably 5 or more, and more specifically, 1 mole per enzyme. ,:! -500 (for example, lysozyme), or 30-3000 (for example, lipase).

When microparticles coexist in these systems, first, the enzyme and the particles are mixed in an aqueous medium to adsorb the enzyme to the particle surface, and thereafter, a charged group or hydrophobic group is added to the end of one hydrophilic polymer chain. Add a polymer with one or many functional groups and add, if necessary, at room temperature, for example, cooling (by number) By mixing and stirring below, the surface of the microparticles bound by the enzyme can be further coated with the polymer. When the enzyme is to be carried on the particles, for example, a method in which a mercapto group is introduced into the enzyme by a general method, if necessary, and binding to a metal surface, for example, can be selected. The ratio of particles to enzyme is also usually determined by conducting small experiments, but the ratio of enzyme to microparticles (by weight) can be used at a ratio of 0.0001: 10000 to 10000: 0.0001, and 1: 10000 to 10000: 1 is more preferable For efficient use, it is also preferable that the ratio be about 1: 1000 to 1000: 1. More specifically, the ratio of enzyme (molecule) to colloidal gold (number) is 0.01: 1 to 100: 1 and, particularly, 20: 1 to: 100: 1. be able to.

The thus obtained enzyme complex comprising the enzyme and the specific polymer, or the enzyme complex in which the enzyme and the specific polymer are bound to the surface is, as specifically described in the following Examples, for example, Strong thermal stability, maintaining the enzyme activity at least 50% even after repeating the cycle of raising the temperature to 70 ° C, holding for 10 minutes, cooling, and raising the temperature again several times. Is shown. Brief Description of Drawings

Figure 1 is a graph showing the transition of enzyme activity after PEG-PMAMA block copolymer and lysozyme were mixed at various mixing ratios and the temperature was repeatedly increased. Symbols in the figure: Squares indicate systems containing no polymer (control), circles indicate systems containing polymers equal in weight to enzyme, and triangles indicate systems containing polymer twice as much as enzyme. , And the inverted triangle indicates the results for a system containing 10 times the weight of the polymer relative to the enzyme.

Figure 2 shows various mixing ratios of PEG-PEAMA block copolymer and lysozyme. 5 is a graph showing the transition of the enzyme activity after mixing and heating. Symbols in the figure: Squares indicate systems containing no polymer (control), diamonds indicate systems containing 1 / 10th the weight of the polymer relative to the enzyme, and white circles indicate an equal weight relative to the enzyme. The solid circles show the results for the system containing twice the weight of the polymer with respect to the enzyme, and the triangles show the results for the system containing 10 times the weight of the polymer with respect to the enzyme.

FIG. 3 is a graph showing changes in enzyme activity after PNIPAM and lysozyme were mixed at various mixing ratios and the temperature was repeatedly increased. Symbols in the figure: Squares indicate systems containing no polymer (control), diamonds indicate systems containing 1 / 10th the weight of the polymer relative to the enzyme, and white circles indicate the same for the enzyme. The system containing the polymer by weight, the closed circles show the results of the system containing 2 times the weight of the polymer relative to the enzyme, and the triangles show the results of the system containing the polymer 10 times the weight of the enzyme. FIG. 4 is a graph showing the transition of the enzyme activity after mixing the PEG-PNIPAM graft copolymer and lysozyme at various mixing ratios and repeatedly raising the temperature. Symbols in the figure: Squares indicate systems without polymer (control), solid diamonds indicate systems containing 1 / 10th the weight of polymer to enzyme, and open circles indicate system for enzyme. A system containing an equal weight of polymer, a black circle represents a system containing a polymer twice as much as the enzyme, a white diamond represents a system containing a polymer three times the weight of the enzyme, and a black reverse. The triangles show the results for the system containing 5 times the weight of the polymer with respect to the enzyme, and the open triangles show the results for the system containing 10 times the weight of the polymer with respect to the enzyme.

Figure 5 is a graph showing the change in enzyme activity after repeated heating of a system containing colloidal gold, PEG-PMAMA block copolymer and lysozyme. The Symbols in the figure: squares indicate systems without polymer and colloidal gold (control), open circles indicate systems containing enzymes and polymers (comparison), and solid circles indicate enzymes, polymers and colloids The result of the system containing metal mold is shown. BEST MODE FOR CARRYING OUT THE INVENTION Embodiment 1

In this example, the polymer (PEG-PMAMA)

CH ₃ OC¾CH ₂ CH ₂ O— (CH ₂ CH ₂ O) _n — (CH ₂ C (CH ₃ )) _m — H

COOCH ₂ CH ₂ N (CH3) 2 (obtained according to the method described in Y. Nagasaki et al., Macromol. Chem. Rapid Commun. 1997, 18, 927. PEG Mn = 4,000 g / mol, PMAMA This is an example using (polymethacrylic acid (2-N, N-dimethylaminoethyl)) Mn = 4,000).

Dissolve 28.6 mg of lysozyme and 28.6 mg of the polymer synthesized above in 50 mL of phosphate buffer (pH 7.0, 50 mM), dilute 40-fold, and mix in 1.5 mL portions. This was used as an enzyme solution. In addition, melted 16. lmg of Micrococcus luteus (Sigma, ATCC No. 4698) in 100 mL of buffer? Then, a substrate solution was prepared.

First, 2.8 mL of the substrate solution was added to the quartz cell and held in a cell holder with the temperature set at 37 ° C. There, the enzyme solution kept at 70 ° C for 10 minutes was cooled to 37 ° C, 0.2 mL was added, and the change in absorbance was measured for 10 minutes. Figure 1 shows the relationship between the enzyme activity obtained from the results of the absorbance measurement and the number of times of temperature rise. When PEG-PMAMA is mixed more than 1x, the activity is completely reduced even when the temperature is increased to 70 ° C. And maintained high enzyme activity.

Example 2:

The procedure described in Example 1 was repeated, except that PEG-PEAMA (PEG segment molecular weight 5, OOOi PEAMA segment molecular weight 5,000, synthesized with reference to the above-mentioned paper) was used instead of PEG-PAMA. Figure 2 shows the relationship between the enzyme activity obtained from the absorbance measurement results and the number of heating. When PEG-PEAMA was mixed twice or more, the activity did not decrease at all even when the temperature was raised to 70 ° C, indicating that high enzyme activity was maintained.

Example 3:

The procedure described in Example 1 was repeated except that PNIPAM (molecular weight: 20,000, synthesized by radical polymerization) was used instead of PEG-PAMA. Figure 3 shows the relationship between the enzyme activity determined from the results of the absorbance measurement and the number of times of temperature rise. When PNIPAM was mixed in an equivalent amount or a double amount, the effect of improving heat resistance was shown. Example 4:

The procedure described in Example 1 was repeated except that PNIPAM-PEG graft polymer (molecular weight 60,000, PEG chain 2,000, synthesized by radical polymerization) was used instead of PEG-PAMA. Fig. 4 shows the relationship between the enzyme activity obtained from the results of the absorbance measurement and the number of heating cycles. When PNIPAM-PEG was mixed in an equivalent amount or more, the effect of improving heat resistance was shown.

Example 5:

In this example, colloidal gold (gold colloid) was added to the enzyme solution, and then a polymer (PEG-PMAMA: polymethacrylic acid (2-N, N-dimethylaminoethyl)) Mn = 4, 00). Lino ヽ⁰ -ze 6. O mg (0.1 l mo 1) was placed in a volumetric flask and adjusted to 100 mL with a phosphate buffer (pH 7.0, 50 mM). Each lmL was collected, 5mL of gold colloid solution (BBI, 10nm) was added, and the mixture was allowed to stand at 4 ° C for 10 minutes. Next, 28.6 mg (4 symbol) of the polymer was dissolved in each, and a solution was added. The solution thus obtained was used as an enzyme solution. Further, a process of mixing 2.0 mL of a buffer having 0.5 mM p-NPP as a substrate, heating to 58 ° C for 10 minutes, cooling to 25 ° C for 5 minutes, and maintaining the temperature was repeated. Figure 5 shows the results of measuring the absorbance at each step. Enzyme activity did not decrease significantly after repeated heating of the system containing colloidal gold, PEG-PMAMA block copolymer and lysozyme.

Industrial applicability

The enzyme complex that can be provided according to the present invention exhibits remarkably high thermal stability, and thus can be used in the manufacturing industry of industrially applicable enzyme catalysts and in the processing of various compounds and materials using the enzyme catalysts.

Claims

The scope of the claims

1. An enzyme complex in which a polymer having one or many charged or hydrophobic groups at the end of a hydrophilic polymer chain is non-covalently bound to an enzyme.

2. The hydrophilic polymer chain is composed of poly (ethylene glycol), poly (2-hydroxyl methacrylate), poly (2-hydroxyl acetylate), poly (bul alcohol), poly (acrylamide). , Poly (methacrylamide), poly (N, N-dimethylacrylamide), poly (N, N-dimethylmethacrylamide), poly (vinylpyrrolidone), agarose and dextran 2. The enzyme conjugate according to claim 1, which is derived from a polymer.

3. The hydrophilic polymer chain is composed of poly (ethylene glycol), poly (2-hydroxyl methacrylate), poly (2-hydroxyl acetylate), poly (bul alcohol), poly (atarylamide) Selected from the group consisting of poly (methacrylamide), poly (N, N-dimethylacrylamide), poly (N, N-dimethylmethacrylamide), poly (vinylpyrrolidone), agarose and dextran. Derived from a polymer, and the charged group is derived from a low-molecular-weight amine compound containing one or two primary, secondary or tertiary amines in the molecule, or repeating a primary, secondary or tertiary amine. 2. The enzyme complex according to claim 1, which is derived from a polymer chain containing one or two kinds in the side chain or main chain of the unit.

4. The hydrophilic polymer chain is derived from poly (ethylene glycol) and the chargeable group is a primary, secondary or tertiary amine with a main or side chain of a repeating unit. 2. The enzyme conjugate according to claim 1, wherein the enzyme conjugate is derived from a polymer chain containing one or two kinds thereof.

5. A polymer having one or many charged groups at the end of a hydrophilic polymer chain is represented by the formula (I):

R ^a -L! -(CH ₂ CH ₂ 0) _n -L ₂ -X (I) In the above formula, ^Ra is hydrogen, methyl, aldehyde, amino, carboxyl, maleimide, p-toluenesulfonyl Group or vinylsulfonyl group,

1 ^ ₂ independently represents a valence bond or a linker; n is an integer from 2 to 20;

X is N- or N, N-mono or di-CL—C ₃ alkyl-substituted amino C—Ci ₂ alkyl, oligo or poly (2 to 20,000) repeating unit N- also properly N, N- mono- also properly is di C one C ₃ alkyl-substituted § Mi Noechirume Taata Li rate) chain, polyethyleneimine Mi down chain, a group selected from the group consisting of scan Perujimin chain and cadaverine chain Or an oligo or polymer chain segment,

The enzyme complex according to claim 1, which is represented by:

6. The enzyme complex according to claim 1, wherein the hydrophilic polymer chain is derived from poly (ethylene glycol), and the hydrophobic group is derived from poly (lactic acid).

7. A polymer having one or more chargeable groups at one end of a hydrophilic polymer chain has the formula:

CH ₃ OCH ₂ CH ₂ CH ₂ 0- (CH ₂ CH ₂₀ ) _n- (CH ₂ C (CH ₃ 》 H

COOCH ₂ CH ₂ N (R) ₂ In the above formula, m and n are each independently an integer of 2 to 20,

R is a C 1 -C ₃ alkyl group,

The enzyme complex according to claim 1, which is represented by:

8. A polymer having one or many charged groups at the end of a hydrophilic polymer chain has the formula:

In the above formula, m, n, and x are each independently an integer of ² to 20, 000, R ¹ is hydrogen or a methyl group, and ¹² is 〇 ₁ —〇 ₃ alkyl group. Ari, R ³ is hydrogen or C! Is an C ₅ alkyl group,

2. The enzyme complex according to claim 1, which is:

9. An enzyme complex in which a polymer having one or many charged or hydrophobic groups at the end of a hydrophilic polymer chain and an enzyme are non-covalently bound on the surface of water-insoluble microparticles.

10. Microparticles from colloidal gold, colloidal silver, colloidal aluminum oxide, colloidal silica, colloidal titanium oxide, micromagnetic steel particles, and colloidal semiconductor The enzyme complex according to claim 9, which is selected from the group consisting of:

1 1. When the hydrophilic polymer chain is poly (ethylene glycol), poly (meth Poly (N-N-N-dimethylacrylamide) Poly (Butyl alcohol), Poly (Butyl alcohol), Poly (Atharylamide), Poly (Metharylamide), Poly (N-N-dimethylacrylamide) 10. The enzyme complex according to claim 9, which is derived from a polymer selected from the group consisting of poly (N, N-dimethylmethacrylamide), poly (vinylpyrrolidone), agarose and dextran.

1 2. The hydrophilic polymer chain is composed of poly (ethylene glycol), poly (2-hydroxyl methacrylate), poly (2-hydroxyl acetylate) poly (vinyl alcohol), poly (atarylamide), Polymers selected from the group consisting of poly (methacrylamide), poly (N, N-dimethylacrylamide), poly (N, N-dimethylmethacryloleamide), poly (vinylpyrrolidone), agarose and dextran And the chargeable group is derived from a low-molecular-weight amine compound containing one or two primary, secondary or tertiary amines in the molecule, or a repeating unit of a primary, secondary or tertiary amine. The enzyme complex according to claim 9, which is derived from a polymer chain containing one or two kinds in the side chain or main chain of the enzyme.

13. A polymer having one or many charged groups at the terminal of a hydrophilic polymer chain is represented by the formula (I):

R ³ -L! _{_{- (CH 2 CH 2 O)}} n - L 2 - in X (I) above formula, R ^a is hydrogen, methylation group, aldehyde group, amino group, carboxyl group, maleimidyl de group, p- toluene A sulfonyl group or a vinyl snolephonyl group,

And L. Represents independently a valence bond or a linker; n is an integer from 2 to 20, 00 00, and

X is, N- Moshiku is N, N- mono- be properly di one C i-C ₃ alkyl substituted amino C i-C ₂ alkyl, having 2 to repeating units 2 0 0 0 0 oligo or poly (N- or N, N-mono- or di-C-Cg alkyl-substituted aminoethylmethatalylate) a group selected from the group consisting of a chain, a polyethyleneimine chain, a sperdimine chain and a cadaverine chain; An oligo or polymer chain segment,

The enzyme complex according to claim 1, which is represented by:

14. The microparticles are suspended in an aqueous solution in which an enzyme is dissolved and bound on the microparticles, and then the suspension obtained in this manner is charged with a charged group or a terminal at the end of the hydrophilic polymer chain. 10. The enzyme complex according to claim 9, which is obtained by adding a polymer having one or many hydrophobic groups and incubating the mixture under conditions sufficient to form an enzyme complex.