JP5867248B2 - Enzyme complex - Google Patents

Description

  The present invention relates to an enzyme complex, and more particularly to a complex of an enzyme that exhibits a function by heat and magnetic particles.

  In recent years, research on proteins has been conducted in many fields spanning science, agriculture, medicine, pharmacy, engineering, and information science. Among these, thermophilic bacterium-derived enzymes that are activated in a high temperature region are attracting attention particularly in the medical field, biotechnology field, environmental field, and the like. However, since an enzyme derived from a thermophilic bacterium has a high optimum reaction temperature, the reaction temperature must be increased, and when used in a biological system, sufficient attention must be paid to its safety.

  On the other hand, various protein hybrid materials in which biomaterials are bound to proteins have been proposed. The protein hybrid material is a complex in which a protein and a substance other than the protein, such as a synthetic polymer, are chemically bound. This protein hybrid material is mainly used for controlling the protein function, and as the material for controlling the protein function, a fine particle material mainly utilizing nanotechnology is used (for example, see Non-Patent Document 1). .

In addition, recent advances in instrumental analysis have made it possible to accurately grasp the structure and characteristics of submicron particles, and therefore, protein hybrid microparticles are being developed for various purposes (for example, Non-patent document 2).
The field that attracts attention is the control of protein function by external stimuli, and the method of controlling the “place”, “time”, and “strength” where the protein exhibits its function by the external environment such as heat and light. It is. Among them, a magnetic hybrid material that is hybridized using a magnetic substance and a protein such as an enzyme or a physiologically active substance has attracted attention as a material that can be easily used to control the function of the protein by an external response because of its magnetic properties. For example, there is a study that uses ferrimagnetic magnetite fine particles as a drug carrier and magnetically controls the induction of a drug to the affected area from the outside (see, for example, Non-Patent Document 3).

Dunnill, P. and Lilly, M.D., "Purification of enzyme using magnetic bio-affinity materials", Biotechnol. Bioeng., 16, P.987-990, 1974 By Haruma Kawaguchi, “Bioaffinity microparticles”, SEN’I GAKKAISHI (Fiber and Industry), Vol.60, No.7, 2004 Akihiko Kondo, Noriyuki Ohnishi, "Development of Thermo-Responsive Magnetic Nano-Particles and Their Application to Biotechnology", PHARM TECH JAPAN, Vol. 19, No. 10 (2003), P.1753-1762

As described above, various applications are expected in protein magnetic hybrid materials, and a new enzyme activity functional control technique using magnetic particles is desired.
Therefore, the present invention uses a hybrid material in which magnetic particles and an enzyme are combined, and further controls the function of enzyme activity of the new magnetic particles by utilizing a heat generation phenomenon caused by applying magnetic energy. Objective.

  The present inventors diligently studied to solve the above problems. As a result, the present inventors have found that an enzyme complex in which an enzyme derived from a thermophilic bacterium and magnetic particles are bound can activate the enzyme even in a low temperature environment by applying magnetic energy.

That is, the present invention is achieved by the following (1) to (7).
(1) An enzyme complex comprising magnetic particles and an enzyme derived from a thermophilic bacterium, and having a temperature responsive function.
(2) The enzyme complex according to (1) above, wherein the magnetic particles are coated with an organic polymer having a carboxyl group, and the carboxyl group and the amino group or hydroxyl group of the enzyme are bonded. body.
(4) The enzyme complex according to any one of (1) to (3) above, wherein the magnetic particles are stimuli-responsive magnetic particles that aggregate by applying a stimulus.
(5) A method for activating the enzyme of the enzyme complex according to any one of (1) to (4) above,
A method for activating an enzyme, comprising activating the enzyme by applying magnetic energy to a solution in which the enzyme complex is dissolved.
(6) The method for activating an enzyme according to (5), wherein the magnetic energy is at least one selected from the group consisting of a magnetic field and an electromagnetic wave.
(7) A cleaning device for a medical instrument, comprising the enzyme complex according to any one of (1) to (4) above and a magnetism generator.
(8) The cleaning apparatus according to (7), wherein the medical instrument is at least one selected from the group consisting of an endoscopic surgical instrument, an invasive surgical instrument, and a suction catheter.

According to the present invention, by applying magnetic energy to the enzyme complex, the surface temperature of the magnetic particles is increased, and the temperature in the vicinity of the magnetic particles is increased to the optimum temperature of the enzyme, so that the overall temperature of the solution is not increased. Both can activate an enzyme derived from a thermophilic bacterium bound to the surface of magnetic particles.
Therefore, it can be effectively used for cleaning malignant proteins such as abnormal prions attached to instruments that cannot be subjected to high temperatures, such as endoscopic surgical instruments.

It is a graph which shows the result of the calibration curve measurement of a substrate decomposition reaction. (A) is a perspective view of a magnetic generator (coil), (B) is sectional drawing of the coil wire of (A). (A) is a schematic diagram of a heat generation evaluation experimental device of Therma-Max (registered trademark), and (B) is a usage form in which a heat insulating material is inserted into a magnetic generator of the heat generation evaluation experimental device of (A). It is a perspective view shown. It is a graph which shows the heat_generation | fever characteristic in each magnetic field strength of Thermo-Max (trademark). (A) is a schematic view of a Thermo-Max (registered trademark) exothermic evaluation experimental apparatus in low-temperature measurement, and (B) is a method for inserting water into a magnetic generator of the exothermic evaluation experimental apparatus of (A). It is a perspective view which shows the usage pattern used circulating. It is a graph which shows the measurement result of the enzyme activity of Tk-subtilisin.Therma-Max (trademark) LC Carboxylic acid complex in 4 degreeC. It is a graph which shows the measurement result of the enzyme activity of Tk-subtilisin * Therma-Max (trademark) LC Carboxylic acid complex in 25 degreeC. It is a graph which shows the measurement result of the enzyme activity of Tk-subtilisin * Therma-Max (trademark) LC Carboxylic acid complex in each temperature.

  The enzyme complex of the present invention comprises magnetic particles and an enzyme derived from a thermophilic bacterium, and has a temperature responsive function. The enzyme complex of the present invention may contain components other than the magnetic particles and the enzyme derived from thermophilic bacteria, as long as the effects of the present invention are not significantly impaired.

(Magnetic particles)
The magnetic particles used in the present invention may be particles composed of iron oxide or ferrite, and are composed of iron oxide, ferrite, or magnetite and other inorganic substances and organic substances such as particles produced from polyhydric alcohol and magnetite. Particles may be used.
The magnetic particles can be produced, for example, by a method disclosed in JP-T-2002-517085. That is, an aqueous solution containing an iron (II) salt or an iron (II) salt and a metal (II) salt is placed in an oxidation state necessary for forming a magnetic oxide, and the pH of the aqueous solution is maintained at 7 or more. Thus, iron oxide or ferrite magnetic particles are formed. It can also be produced by mixing an aqueous solution containing a metal (II) salt and an aqueous solution containing an iron (III) salt under alkaline conditions.

  Alternatively, the magnetic particles can be produced from polyhydric alcohol and magnetite. The polyhydric alcohol can be used without particular limitation as long as it is an alcohol structure having at least two hydroxyl groups in the structural unit and capable of binding to iron ions. For example, dextran, polyvinyl alcohol, mannitol, sorbitol, cyclodextrin and the like can be mentioned. For example, JP 2005-082538 A discloses a method for producing magnetic particles using dextran, which can also be produced by this method. Moreover, the compound which has an epoxy group and forms a polyhydric alcohol structure after ring-opening like a glycidyl methacrylate polymer can also be used.

In order for the magnetic particles to have good dispersibility, it is preferable that the average particle diameter of the particles before being aggregated by applying magnetic energy is 1 to 1000 nm, and to have good heat generation characteristics. 1 to 500 nm is more preferable. In particular, the average particle diameter of the particles is more preferably 3 to 200 nm, and particularly preferably 5 to 50 nm, from the viewpoint of improving the reactivity between the magnetic particles and the enzyme and the heat generation efficiency. Specifically, when the magnetic particles are particles made of magnetite, heat can be generated favorably in an alternating magnetic field of 100 kHz to 1 MHz when the average particle diameter is about 15 to 20 nm. As a device capable of performing such particle size measurement, there are a particle size / particle size distribution measuring device (Zeta Sizer Nano ZS, manufactured by Malvern) and a transmission electron microscope (Hitachi Transmission Electron Microscope HT7700, manufactured by Hitachi High-Technologies Corporation). Can be mentioned. These magnetic particles may form aggregates in the solution.
Examples of commercially available magnetic particles that can be used in the present invention include Dynabeads (registered trademark), nanomag (registered trademark), deStars, and MACS (registered trademark).

  The magnetic particles are preferably modified with a polymer or the like from the viewpoint of protecting the particles from the external environment or functionalizing the particle surface. As the protective agent to be used, various functional materials such as polymers such as PVA and dextran, and inorganic materials such as silica are used.

Examples of magnetic particles whose particle surfaces are protected and functionalized include, for example, “Therma-Max (registered trademark)” (hereinafter also referred to as “Thermamax”), “Therma-Max” (registered trademark) of JNC Corporation. (Trademark) LC Carboxylic acid "(hereinafter sometimes referred to as" TM-LC "). In “Therma-Max (registered trademark)”, a polymer of N-isopropylacrylamide (hereinafter sometimes referred to as “NIPAM”) is modified on the surface of iron oxide magnetic particles.
In the present specification, “thermamax” simply indicates magnetic particles, and “thermamax solution” and “thermamax undiluted solution” indicate that the magnetic particles contain a buffer or the like. .
The NIPAM aqueous polymer solution can control the temperature at which it dissolves and precipitates (lower critical solution temperature) depending on the degree of polymerization of the polymer, and has a lower critical solution temperature around 32 ° C. However, at a temperature higher than this, it aggregates and can be easily recovered magnetically.
Therefore, the thermamax reversibly changes between both agglomerated and dispersed states at a solution temperature of 32 ° C. due to NIPAM coating the particle surface. Therefore, in a dispersion state of less than 32 ° C. where the particles are uniformly dispersed in the solution, magnetic separation is not easy, but when the solution temperature is raised to 32 ° C. or more, the magnetic particles begin to form aggregates, and magnetic separation Easy to do.

  Since the functionalized magnetic particles can be made into a desired form such as agglomeration by applying a stimulus, applying an external stimulus to the functionalized magnetic particles (stimulus-responsive magnetic particles) Thus, the efficiency of preparation and purification of the enzyme complex can be dramatically increased while maintaining the dispersibility of the particles.

  Examples of the stimulus applied to the stimulus-responsive magnetic particles include a temperature change, a pH change, a light change, an ionic strength change, and the like, and the conditions of each stimulus may be adjusted as appropriate.

(Enzyme from thermophile)
The enzyme derived from a thermophilic bacterium used in the present invention has an enzyme activity value that changes with temperature, has an activity in a high temperature region of 65 ° C. or higher, and is equilibrium, kinetic, or its high temperature. It has the characteristic of being stabilized in both.

  Examples of the enzyme derived from thermophile include protease, amylase, cellulase, esterase, DNA polymerase, nuclease, peroxidase and the like in general names. Specifically, TK-subtilisin, Tk-SP, Tk-GK, Tk-RNaseH, etc. are mentioned.

(Method of binding magnetic particles to thermophilic bacteria-derived enzyme)
The enzyme complex of the present invention is produced by combining the magnetic particles and the enzyme derived from the thermophile.
As a method for producing the enzyme complex of the present invention, a method for binding the magnetic particles and the enzyme may be appropriately selected depending on the surface form of the magnetic particles. Examples of the binding method include a direct method in which an enzyme is directly bound to the surface of the magnetic particle and an indirect binding method in which the enzyme is bound to the surface of the magnetic particle through an organic polymer that coats the surface of the magnetic material.

  In the direct method, an enzyme is directly adsorbed on magnetic particles. For example, a method in which the enzyme adsorbed on the surface of the magnetic particles is cross-linked with glutaraldehyde to coat the surface of the magnetic particles.

  The indirect bonding method is a technique in which a magnetic particle and an enzyme are bonded through an organic polymer coated on the magnetic particle. In order to coat the organic polymer, there are two kinds of methods in which a polymerization reaction is performed on the particle surface or the organic polymer is bonded. Typical examples are coatings with dextran or methacrylic acid polymers. In order to bind the coated magnetic substance to the enzyme, a usual chemical modification means is used. In general, binding to an enzyme complex by an aminosilanized surface or a surface having a carboxyl group activated by carbodiimide is known. These reactions are considered to be milder than the direct binding method, and tend to retain the activity of the bound enzyme as compared to the enzyme prepared by the direct method.

  In the present invention, it is preferable to immobilize the enzyme on the carboxyl group in the organic polymer having a carboxyl group coated on the surface of the thermamax by a condensation reaction using carbodiimide. Carbodiimide is a dehydration condensing agent that causes a dehydration condensation reaction between a carboxyl group and an amino group or a hydroxyl group. By this reaction, an acid amide or ester bond is formed.

(Temperature response function)
The enzyme complex of the present invention thus produced has a temperature responsive function. The temperature responsive function generates heat due to the magnetic energy accumulated in the magnetic particles due to a delay in the magnetization response to the fluctuating magnetic field, and the enzyme fixed to the magnetic particles is heated by the heat generation of the magnetic particles, thereby expressing the enzyme activity. That means.

(Magnetic generator)
A method for imparting magnetic energy to the enzyme complex is not particularly limited, and for example, a magnetic field, an electromagnetic wave, a permanent magnet, or the like can be used. Among these, it is preferable to form a magnetic field using a magnetic generator that generates magnetism with an alternating current because it is easy to handle and the intensity of magnetic force can be easily changed.

  In the case of using an alternating magnetic field, the condition of the magnetic field to be added may be appropriately selected according to the type of enzyme used. For example, the magnetic field strength is preferably 0.1 to 2,000 Oe, 000 Oe is more preferable, and 3-500 Oe is more preferable. By setting it as the said range, the emitted-heat amount of a magnetic particle is controllable. The frequency is preferably 1,000 to 15,000,000 Hz, more preferably 10,000 to 3,000,000 Hz, and even more preferably 100,000 to 1,000,000 Hz. preferable. By setting it as the above range, the influence of dielectric heating can be reduced and only the magnetic particles can generate heat.

  By applying magnetic energy from the outside of the solution to the solution in which the enzyme complex of the present invention described above is dissolved, the magnetic particles of the enzyme complex generate heat. The solution temperature rises. When the temperature rises to the optimum temperature of the enzyme, the enzyme function is expressed. Therefore, the activity of an enzyme having a temperature responsive function can be increased without increasing the temperature of the entire solution. Therefore, for example, the enzyme complex of the present invention can be used even in situations where use in a high temperature region is restricted.

For example, a cleaning instrument for a medical instrument can be provided by including the enzyme complex of the present invention and a magnetic generator.
Endoscopic surgical instruments are susceptible to adhesion of malignant proteins such as denatured prions, but are difficult to disinfect at high temperatures due to the material of the instruments. Since Tk-subtilisin, which is an enzyme derived from a thermophilic bacterium, is an enzyme that degrades prions, if the endoscopic surgical instrument is washed with a magnetic generator using the enzyme complex of the present invention to which Tk-subtilisin is immobilized. Since the activity of Tk-subtilisin can be increased without increasing the temperature of the washing solution, the denatured prion can be reliably decomposed and removed.

  In addition to the endoscopic surgical instruments described above, examples of medical instruments include invasive surgical instruments such as scalpels, scissors, and forceps, instruments that are inserted into the body, such as suction catheters, and resin surgical instruments. can do.

EXAMPLES Next, although an Example demonstrates this invention further in detail, this invention is not limited at all by these examples.
The purified water used in the present invention is ultrapure water having a specific resistance of 18 MΩ · cm or more purified by Direct-Q (trade name) manufactured by Millipore.

<Enzyme Complex: Synthesis of Tk-Satilisin / TM-LC Complex>
An enzyme complex was prepared according to the following protocol using Thermo-Max (registered trademark) LC Carboxylic acid (manufactured by JNC Corporation) as magnetic particles and Tk-satilysin as an enzyme.
[1] TM-LC activation step 1 using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (hereinafter EDC) / N-hydroxysuccinimide (hereinafter NHS) 1) 500 μl TM-LC was dispensed into a microtube, and 19.5 μl of 4M NaCl aqueous solution was added and mixed.
Step 2) After the mixed solution is placed in a thermostatic bath and heated for one minute, the microtube is converted into a magnetic separation device (permanent magnet (neodymium magnet, φ30 × 10 mm (round shape), magnetization direction: thickness direction, surface magnetic flux density: 445 mT)). ) Was allowed to stand for 1 minute, and TM-LC aggregates were magnetically separated to remove the supernatant.
Step 3) 500 μl of 25 mM MES buffer (in 50 mM NaCl, pH 4.75) (hereinafter referred to as Activation Buffer) was added and dispersed in an ice bath.
Step 4) The same operation as in step 2 was performed.
Step 5) 300 μl of Activation Buffer was added to the microtube and dispersed in an ice bath.
Step 6) 2 mg of EDC and 4 mg of NHS were dissolved in 200 μl of Activation Buffer. This adjustment liquid was mixed with the solution of Step 5 and rotated and mixed at room temperature (about 25 ° C.) for 1.5 hours.
Step 7) The same operation as in Step 2 was performed to remove unreacted EDC / NHS.
Step 8) 300 μl of 10 mM acetate buffer (in 50 mM NaCl, pH 5.0) (hereinafter referred to as “Binding Buffer”) was added and dispersed in an ice bath.
Step 9) Steps 7 and 8 were further performed twice.
[2] Immobilization of Tk-subtilisin to activated TM-LC 10) After the operation of step 9, 300 μl of 0.1 mg / ml Tk-subtilisin solution (in 50 mM acetate buffer, pH 5.0) was added to the particles. In addition, the mixture was dispersed in an ice bath, and then rotated and mixed at 4 ° C. for 3 hours.
Step 11) The same operation as in Step 2 was performed to remove unreacted Tk-satilysin. The supernatant after magnetic separation was stored for later protein quantification.
Step 12) 500 μl of 50 mM Tris-HCl buffer (pH 7.5) (hereinafter referred to as “Reaction Stop Buffer”) was added to stop unreacted activating groups. After dispersing in an ice bath, rotary mixing was performed at 4 ° C. for 30 minutes.
Step 13) The same operation as in step 2 was performed.
Step 14) 50 mM acetate buffer (pH 5.0) (hereinafter referred to as acetate buffer) was added and dispersed in an ice bath.
Step 15) Steps 13 and 14 were repeated twice more.
Step 16) The Tk-subtilisin / TM-LC complex solution was stored in a refrigerator in 500 μl of the above acetate buffer.

<Evaluation of enzyme complex>
For the synthesized Tk-subtilisin.TM-LC complex, the iron oxide concentration in the Tk-subtilisin.TM-LC complex solution by ICP measurement, the quantification of the enzyme amount of Tk-subtilisin by the differential method, and the Tk-subtilisin.TM. Three indicators of enzyme activity of the LC complex solution by the azocasein method were measured. From these three measured values, in the Tk-subtilisin / TM-LC complex solution,
i. Enzyme mass per particle (enzyme mass / particle mass),
ii. Enzyme activity value per enzyme (enzyme activity / enzyme mass),
iii. Enzyme activity value per particle (enzyme activity / particle mass)
Was calculated and the Tk-saturisin / TM-LC complex solution was evaluated.

(ICP emission analysis)
First, a calibration curve was prepared by diluting a standard solution of Fe ²⁺ (0, 5, 20 ppm), and based on this, the iron content in the Tk-satilysin · TM-LC complex was determined.

  The amount of iron oxide particles in the Thermamax stock solution and the Tk-satilysin · TM-LC complex solution was evaluated by the amount of iron atoms contained in each solution. The results are shown in Table 1.

  From the results shown in Table 1, the thermamax stock solution, which had iron particles of 4 mg / ml as the Fe concentration, was 1.38 mg as the Fe concentration in the Tk-satilysin · TM-LC complex solution after immobilization. / Ml of iron particles. From this, it was found that the Tk-subtilisin / TM-LC complex solution has about 35% iron particles relative to the Thermamax stock solution.

(Quantitative analysis of Tk-subtilisin in Tk-subtilisin / TM-LC complex)
The amount of Tk-subtilisin immobilized on the magnetic particles was calculated by the difference method. Azocasein was used as a substrate molecule that reacts with the enzyme. Azocasein is decomposed by an enzyme to release a compound containing an azo group. Since the compound containing this azo group has absorption at 440 nm, its absorbance can be quantified by measuring with a UV-vis spectrophotometer (model number V-650, manufactured by JASCO Corporation).
Absorbance measurement with a UV-vis spectrophotometer was performed by making light incident on the sample solution, measuring the transmitted light intensity I, and calculating the absorbance A from the following equation (1).

In addition, since the extinction coefficient of p-nitroanilide generated from the substrate N-succinyl-AAPF-p-nitroanilide is 8900 M ⁻¹ cm ⁻¹ and measurement was performed at a cell length of 1 cm, the molar concentration c is
_{c = A 440 / εd = A} 440/8900
Calculated with

  In order to quantitatively evaluate the concentration of Tk-subtilisin remaining in the supernatant, the enzymatic degradation reaction of Tk-subtilisin at each Tk-subtilisin concentration using azocasein as a substrate was measured, and the prepared calibration curve is shown in FIG.

  From the results of FIG. 1, it can be seen that the substrate degradation reaction of azocasein occurs in proportion to Tk-subtilisin between the measured concentrations, and the peak observed in the vicinity of 440 nm derived from the degradation product is increased. Therefore, the same azocasein substrate decomposition reaction experiment was performed on the Tk-subtilisin solution stock solution used for the enzyme immobilization reaction and the Tk-subtilisin supernatant solution recovered after the completion of the reaction. The Tk-subtilisin concentration was calculated. The results are shown in Table 2 below.

  By taking the difference between the enzyme concentration results calculated in Table 2, the concentration of immobilized Tk-subtilisin was calculated. The results are shown in Table 3 below.

  From the results of Table 3, the enzyme concentration in the Tk-satilysin solution was 107.5 μg / ml, but the enzyme concentration in the Tk-satilysin supernatant solution after immobilization was 5.0 μg / ml. From this, it was confirmed that 95.3% of the enzyme concentration of the stock solution of Tk-subtilisin charged at the time of immobilization was immobilized on the surface of Thermamax. From the particle concentration obtained from the ICP measurement in Table 3, the enzyme amount per particle weight was 3.6 μg / mg.

(Enzyme activity analysis)
Next, the enzyme activity of free Tk-subtilisin not immobilized on the magnetic particles was measured and compared with that at the time of immobilization. N-succinyl-AAPF-p-nitroanilide was used as a substrate for enzyme activity measurement. As shown in the following formula (A), N-succinyl-AAPF-p-nitroanilide has a peptide bond hydrolyzed by a protease to produce p-nitroaniline.
(Formula A)

The degradation substance was quantified by measuring the absorbance of this p-nitroaniline. The decomposition amount of the substrate N-succinyl-AAPF-p-nitroanilide was performed using Tk-subtilisin, and the amount of p-nitroaniline produced was calculated from the Lambert-Beer law by plotting the absorbance at 410 nm. . The enzyme activity was evaluated using an evaluation index called unit (U). In the present invention, one unit is defined as the amount of enzyme that produces 1 μmol of p-nitroanilide per minute.
As a specific experimental operation, 95 μl of 0 to 5 mM N-succinyl-AAPF-p-nitroanilide solution was prepared, and a Tk-subtilisin / TM-LC complex solution having an enzyme concentration of 0.010 mg / ml was prepared for each. 5 μl of each was added, and the enzyme reaction was performed at 20 ° C. for 5 minutes. Thereafter, 10 μl of 5 wt% acetic acid aqueous solution was added to stop the enzyme reaction, and the solution was passed twice through a magnetic separation column (MS Columns, manufactured by Miltenyi Biotech), and then at a wavelength of 410 nm in the supernatant solution. P- produced from N-succinyl-AAPF-p-nitroanilide using a UV-vis spectrophotometer (VARIAN, CARY50Probe) and using an extinction coefficient of 8900 M ⁻¹ cm ^−1. Calculated as the amount of nitroanilide.

  Moreover, the same experiment was conducted using free Tk-subtilisin adjusted to 0.010 mg / ml, which is the same as the enzyme concentration of the immobilized enzyme. The enzyme activity value (unit) was calculated from the obtained absorbance at 410 nm, and the ratio of the enzyme retaining the activity was evaluated by comparing the values.

  From the results of absorption spectra at 410 nm for each substrate concentration, a unit which is an enzyme activity index is calculated, and the degree of activity maintained by the enzyme immobilized on the enzyme complex is shown in Tables 4 and 5. Show.

  The activity retention rate in Table 5 indicates how much activity is retained by comparing the immobilized enzyme with a free enzyme. This was calculated by dividing the immobilized enzyme activity value (U) by the free enzyme activity value (U). From Table 5, an average of about 46% activity is retained. Although this value differs depending on the structure of the enzyme and the content of the amino group, the enzyme immobilization rate is different from that of the conventional carbodiimide method.

From the above results, the amount of enzyme contained in the Tk-satilysin · TM-LC complex solution of the example is
(Particle concentration) × (volume) = 0.010 mg / ml × 5 μl = 0.05 μg = 5 × 10 ⁻⁵ mg
Based on this, the enzyme activity (specific activity) per 1 mg of enzyme was calculated. Usually, when calculating the specific activity of an enzyme, the value at the time of a substrate excess in which the absorbance does not change even when the substrate concentration is increased is used. In this measurement, it can be considered that the absorbance does not increase from the substrate concentration of 5 mM. Therefore, the specific activity of the immobilized enzyme was calculated from the enzyme activity value (U) at this time. The results are shown in Table 6.

  From the results of Table 6, it can be said that even when compared with the enzyme activity value per free enzyme weight not bound to the magnetic particles, the value is not inferior.

<Evaluation of heat generation of magnetic particles by AC magnetic field>
Therma-Max (registered trademark, manufactured by JNC Corporation) was used as magnetic particles, and an AC magnetic field application test was performed in order to evaluate the heat generation behavior of thermamax by the AC magnetic field.

(High-frequency magnetism generator)
[Magnetic generator]
A magnetic generator 10 (manufactured by Samway Corporation) shown in FIGS. 2 (A) and 2 (B) was used. The magnetic generator 10 is configured by winding a coil 11 around the outer periphery of an inner cylinder 13. The coil 11 for generating a high-frequency magnetic field is composed of a copper pipe (coil wire) 11a provided with an insulating coating 11b having an inner diameter of 3 mm, an outer diameter of 4 mm, and a thickness of 0.25 mm. The inner cylinder 13 of the coil 11 is made of Teflon (registered trademark), has an inner diameter of 58 mm, an outer diameter of 70 mm, and a height of 120 mm. The number of turns of the coil 11 is 23 turns. The magnetic field intensity handled in the present invention is expressed as an effective value (Oe-rms). Moreover, in order to suppress the heat_generation | fever of the coil 11, the cooling water controlled by 20 degreeC was poured in the copper pipe of the coil 11 during the measurement.

[Description of other experimental equipment]
(1) High frequency power supply (model T162-6024AHE, manufactured by Samway Corporation)
This is a variable frequency type high frequency power source, and has a frequency band of 100 to 1999 kHz and a maximum output of 1000 W / 50Ω.
(2) Matching box (Samway Corporation)
A coil and a capacitor are built in, and their inductance and capacitance are designed to be variable in conjunction with the attached lever. By adjusting those levers, the resonance point of the experimental coil can be found.
(3) Chiller for matching box (model TBG045 A A, manufactured by ADVANTEC)
(4) Optical fiber type thermometer (model Reflex-4, manufactured by Neooptix)
The measurable temperature range is -80 to 250 degrees. The resolution is 0.1 degree.
(5) Oscilloscope (model TDS1000B, manufactured by Tektronix)
This was used to measure the current value flowing through the high frequency coil.
(6) Rogowski coil (model Pearson, manufactured by Electronics)
When measuring the alternating magnetic field, this coil is used to detect the current flowing in the experimental coil. The electromotive force generated in the Rogowski coil is detected by an oscilloscope, and the converted current value is displayed.

(Heat generation evaluation experiment in the heat insulation state using Thermo-Max)
In order to verify whether the thermox functions as a magnetic heating element, an experiment for evaluating heat generation in an adiabatic state was performed using a heat generation evaluation experimental apparatus shown in FIGS. 3 (A) and 3 (B). The experimental procedure is described below.

[Experimental procedure]
Step 1) 1 ml of the Thermamax solution 17 having a particle concentration of 2 mg / ml is placed in the Eppendorf tube 15.
Step 2) The optical fiber thermometer 19 is put in the middle of the thermax solution 17, the Eppendorf tube 15 is insulated with the foamed polystyrene (heat insulating material) 18, and placed at the center of the high frequency magnetic field generating coil.
Step 3) Monitor the increase in the solution temperature at 1 MHz at each magnetic field strength for 1000 s.

(Evaluation of heat generation characteristics by thermamax AC magnetic field)
FIG. 4 shows the experimental results of evaluating heat generation in the heat insulating state using thermamax. The applied magnetic field strength was 30, 40, and 50 Oe, the horizontal axis represents time, and the vertical axis represents the rising temperature ΔT (° C.).

From the experimental results shown in FIG. 4, it was found that the thermomax solution showed a temperature increase of about 5.5 ° C. in 1000 seconds at 50 Oe and about 4.5 ° C. at 40 Oe (FIG. 4 (a)). ), (B)).
Further, when the intensity of the alternating magnetic field was set to 30 Oe, it was found that the heat generation of the magnetic particles due to the alternating magnetic field and the heat radiation in the entire system were balanced, and almost no temperature increase was observed (FIG. 4C). Therefore, from this result, it was found that by applying an AC magnetic field having an appropriate strength to the therma max, the solution temperature could not be increased and energy could be applied only in the vicinity of the particles.

(Evaluation of thermal response of organic polymer on particle surface by AC magnetic field)
In order to verify whether the NIPAM polymer responds thermally when an AC magnetic field is applied and the thermamax is agglomerated, an AC magnetic field with a magnetic field strength of 26 Oe is applied at a low temperature of 3 ° C and 20 ° C near room temperature. And the average particle size of thermamax immediately after application were measured.
In the present invention, the average particle diameter is a value measured with an apparatus (Zeta Sizer Nano ZS, manufactured by Malvern) based on a dynamic light scattering method (abbreviation of DLS: Dynamic Light Scattering). The application method of the alternating magnetic field at 20 ° C. was performed according to the above [Experimental procedure]. In addition, as shown in FIGS. 5A and 5B, the application of the alternating magnetic field at a low temperature (3 ° C.) is performed through a system that circulates water 21 at 4 ° C. in the magnetic generator 10 of the heat generation evaluation experimental device. Using. The results are shown in Table 7.

  From the results shown in Table 7, particle aggregation as seen when the external temperature was increased was observed even when an alternating magnetic field was applied. From these results, it was confirmed that the NIPAM polymer on the particle surface thermally responded and caused aggregation due to the exothermic phenomenon of iron oxide that occurred when an alternating magnetic field was applied. In other words, it was found that the enzyme activity can be controlled by applying energy to the enzyme immobilized on the magnetic particles by turning the AC magnetic field on and off.

<Evaluation of activity of immobilized enzyme by AC magnetic field>
In order to investigate the influence of the application of an alternating magnetic field on the activity of the immobilized enzyme, the following experiment was conducted.

(Effect of temperature change on enzyme activity of immobilized enzyme)
The Tk-subtilisin / TM-LC complex at each temperature was measured, and the change in the enzyme activity value due to the external temperature was measured.
First, N-succinyl-AAPF-p-nitroanilide was diluted with DMSO, and 10 μl of a substrate solution adjusted to 200 mM was added to 823 μl of enzyme activity buffer (50 mM Tris-HCl, pH 8.0, 1 mM CaCl ₂ ) and 166 μl of the Tk-subtilisin / TM-LC complex solution were added to make a total volume of 1 ml. This solution was heated using an aluminum block thermostat (MG-2200) so as to be constant at 4 ° C., 25 ° C., 45 ° C., and 65 ° C., and reacted for 10 min. Thereafter, 100 μl of 5 wt% acetic acid aqueous solution was added to stop the enzyme reaction, and the magnetic particles were completely removed by passing twice through a magnetic separation column (MS Columns, manufactured by Miltenyi Biotech). The absorbance change (410 nm) of the supernatant solution was measured and evaluated using a UV-vis spectrophotometer (V-650, manufactured by JASCO Corporation).

In addition, since both the substrate N-succinyl-AAPF-p-nitroanilide and thermamax have absorption in a low wavelength region, it is difficult to evaluate a wavelength region of 400 nm or less. Therefore, as a control, a sample in which the substrate N-succinyl-AAPF-p-nitroanilide was added to a commercially available thermax solution was prepared, and evaluation was performed by subtracting this spectrum as a background. Details of this background measurement will be described below.
N-succinyl-AAPF-p-nitroanilide diluted with DMSO and adjusted to 200 mM 10 μl of substrate solution 823 μl of enzyme activity buffer to a final concentration of 1 mM (50 mM Tris-HCl, pH 8.0, 1 mM CaCl ₂ ) Then, 66 μl of commercially available thermomax (stock solution, 4 mg / ml) was added to make a total volume of 1 ml. Then, an alternating magnetic field was applied to this solution at a solution temperature of 25 ° C., a magnetic field strength of 40 Oe, and 1 MHz for 10 minutes. The other AC magnetic field conditions were set the same as above. Thereafter, 100 μl of a 5 wt% acetic acid aqueous solution was added to stop the enzyme reaction, and the solution was passed through a magnetic column twice. Then, the absorbance change at a wavelength of 410 nm was measured with a UV-vis spectrophotometer (CARY50 Probe, manufactured by VARIAN). ) Was used for measurement and evaluation.

(Evaluation of the effect on immobilized enzyme activity by application of an alternating magnetic field at a solution temperature of 4 ° C)
Using the cooling system shown in FIG. 5, the influence on the immobilized enzyme activity by application of an alternating magnetic field at a solution temperature of 4 ° C. was evaluated. As a sample, 1 ml of an enzyme-immobilized enzyme solution prepared in the same manner as described above (influence on the enzyme activity of the immobilized enzyme) was applied, and an alternating magnetic field was applied for 10 minutes at 4 ° C., a magnetic field strength of 40 Oe, and 1 MHz. The other AC magnetic field conditions were the same as described above. After applying the alternating magnetic field, 100 μl of 5 wt% acetic acid aqueous solution was added to stop the enzyme reaction, and the solution was passed twice through a magnetic separation column (MS Columns, manufactured by Miltenyi Biotech Co., Ltd.). The change in absorbance was measured and evaluated using a UV-vis spectrophotometer (CARY50 Probe, manufactured by VARIAN). And the obtained result was compared with the result in 4 degreeC in (the influence on the enzyme activity of the fixed enzyme by a temperature change). FIG. 6 shows changes in enzyme activity at 4 ° C. when the alternating magnetic field is turned on / off.

When both were compared by the absorbance at a wavelength of 410 nm, the amount of p-nitroaniline, which is a substrate decomposition product, at a solution temperature of 4 ° C. was higher than that of an AC magnetic field (FIG. 6B). It was confirmed that the magnetic field applied (FIG. 6A) increased about 1.2 times.
From this result, activation of the enzyme function by an alternating magnetic field was shown in a low temperature (4 ° C.) region, and it was found that the increase in activity was about 1.2 times.

(Evaluation of the effect on immobilized enzyme activity by application of an alternating magnetic field at a solution temperature of 25 ° C.)
Similarly, for the solutions prepared in the above (influence on enzyme activity of immobilized enzyme due to temperature change), the influence on immobilized enzyme activity due to application of an alternating magnetic field at a solution temperature of 25 ° C. was evaluated. An alternating magnetic field having a magnetic field strength of 40 Oe was applied at 25 ° C. for 10 minutes. After applying an alternating magnetic field, 100 μl of 5 wt% acetic acid aqueous solution was added to stop the enzyme reaction, and the solution was passed twice through a magnetic separation column (MS Columns, manufactured by Miltenyi Biotech Co., Ltd.). The change in absorbance was measured and evaluated using a UV-vis spectrophotometer (CARY50 Probe, manufactured by VARIAN). And the obtained result was compared with the result in 25 degreeC in the above (influence on the enzyme activity of the immobilized enzyme by temperature change). FIG. 7 shows changes in enzyme activity at 25 ° C. when the alternating magnetic field is turned on and off.

When both were compared by the absorbance at a wavelength of 410 nm, the amount of p-nitroaniline that is a decomposition product of the substrate at a solution temperature of 25 ° C. was higher than that of an AC magnetic field (FIG. 7B). It was confirmed that the magnetic field applied (FIG. 7A) increased about 1.6 times.
From this result, it is considered that a significant difference was observed in the vicinity of the immobilized enzyme due to application of an alternating magnetic field.

(Evaluation of immobilized enzyme activity by turning on / off alternating magnetic field application)
From the results obtained above (Evaluation of influence on immobilized enzyme activity by application of alternating magnetic field at solution temperature of 4 ° C.) and above (Evaluation of influence of immobilized enzyme activity by application of alternating magnetic field at solution temperature of 25 ° C.) The extent of the temperature increase in the vicinity of the enzyme caused by the AC magnetic field was verified by comparing with the activity measurement measured without applying the AC magnetic field. The result of measurement without applying an alternating magnetic field is the result of the measurement of the enzyme activity of the immobilized enzyme as described above (influence on the enzyme activity of the immobilized enzyme due to temperature change). 4 ° C. and 25 ° C. performed in the above (Evaluation of influence on immobilized enzyme activity due to application of alternating magnetic field at solution temperature of 25 ° C.) Was used and evaluated. The result is shown in FIG.

  From the result of FIG. 8, it can be seen that the enzyme activity is higher when the AC magnetic field is applied (FIG. 8A) than when the AC magnetic field is not applied (FIG. 8B). The result of enzyme activity with a magnetic field was about 1.2 times that in the absence of a magnetic field, but this was found to correspond to the enzyme activity value at about 5 ° C. in the absence of a magnetic field. On the other hand, at 25 ° C., the enzyme activity value with a magnetic field is 1.6 times that at no magnetic field, which is equivalent to the enzyme activity at 35 ° C., which is 10 ° C. higher than without magnetic field. It was. Therefore, it was confirmed that by applying an alternating magnetic field in a higher temperature range, a function with good magnetic field responsiveness can be obtained.

DESCRIPTION OF SYMBOLS 10 Magnetic generator 11 Coil 13 Inner cylinder 15 Eppendorf tube 17 Thermamax solution 18 Thermal insulation material 19 Optical fiber thermometer

Claims (10)

It consists of stimuli-responsive magnetic particles that reversibly change between agglomeration and dispersion states due to temperature changes, and an enzyme derived from thermophilic bacteria, and has a temperature-responsive function and does not contain precious metals. Enzyme complex.   The enzyme complex according to claim 1, wherein the magnetic particles are coated with an organic polymer having a carboxyl group, and the carboxyl group and an amino group or a hydroxyl group of the enzyme are bonded to each other.   The enzyme complex according to claim 1 or 2, wherein the magnetic particles are magnetic particles having an average particle diameter of 1 to 1000 nm. A method for activating an enzyme of an enzyme complex comprising:
The enzyme complex is an enzyme complex having a temperature-responsive function composed of stimuli-responsive magnetic particles that reversibly change between an aggregation state and a dispersion state due to a temperature change and an enzyme derived from a thermophilic bacterium,
A method for activating an enzyme, wherein the enzyme is activated by applying an alternating magnetic field to a solution in which the enzyme complex is dissolved. The method for activating an enzyme according to claim 4 , wherein the magnetic particles are coated with an organic polymer having a carboxyl group, and the carboxyl group and the amino group or hydroxyl group of the enzyme are bonded to each other. 6. The enzyme activation method according to claim 4 , wherein the magnetic particles are magnetic particles having an average particle diameter of 1 to 1000 nm. It is provided with an enzyme complex having a temperature-responsive function composed of stimuli-responsive magnetic particles that reversibly change between agglomeration and dispersion states due to temperature changes and an enzyme derived from a thermophilic bacterium, and a magnetic generator. A medical device cleaning apparatus characterized by the above. The cleaning apparatus according to claim 7 , wherein the medical instrument is at least one selected from the group consisting of an endoscopic surgical instrument, an invasive surgical instrument, and a suction catheter. The cleaning apparatus according to claim 7 or 8 , wherein the magnetic particles are coated with an organic polymer having a carboxyl group, and the carboxyl group is bonded to an amino group or a hydroxyl group of the enzyme. . The cleaning device according to any one of claims 7 to 9 , wherein the magnetic particles are magnetic particles having an average particle diameter of 1 to 1000 nm.

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