JP2009005669A - Immobilized enzyme nanofiber, method for producing the same, and reaction apparatus using the nanofiber - Google Patents

Abstract

The present invention provides an immobilized enzyme nanofiber that hardly causes enzyme deactivation during production, has high enzyme activity, is stable for a long period of time, a method for producing the same, and a reaction apparatus using the nanofiber.
An immobilized enzyme nanofiber 14 can be produced using a method having a step of spinning a raw material solution containing a polymer compound and an enzyme by an electrospinning method, and the diameter spun by the electrospinning method. Enzymes such as lipase are immobilized in a fibrous polymer matrix of 100 nm to 3 μm. Moreover, the reaction apparatus uses the nonwoven fabric formed with the immobilized enzyme nanofiber 14 as an immobilized enzyme carrier, and thus the reaction efficiency is high and the economic efficiency can be improved as compared with the batch method.
[Selection] Figure 1

Description

The present invention relates to an immobilized enzyme nanofiber, a production method thereof, and a reaction apparatus using the nanofiber.

Enzymes are used for production of raw materials and raw material processing in brewing, fermentation of cheese, textile industry, leather factory, food industry, cosmetics industry, pharmaceutical industry, and the like. The enzyme reaction proceeds with high yield and high selectivity under mild reaction conditions, and hardly produces by-products. Therefore, it is expected to be applied to a wide range of fields as a chemical process with high efficiency and low environmental impact. In recent years, biosensors using high substrate specificity have also been applied.
Conventionally, batch method (batch method) was mainly used for substance production using enzyme reaction. However, since this method can be used only once, it is economically inefficient. Application to the continuous production process is under consideration. When the enzyme is applied to a continuous production process, an immobilized enzyme in which the enzyme is immobilized on a carrier and insolubilized in a reaction solvent is used. The carrier and the immobilization method used for immobilizing the enzyme are appropriately selected from the viewpoints of denaturation and prevention of enzyme loss, securing the contact area between the reaction substrate and the enzyme, and the like.

Conventionally used enzyme immobilization methods include a carrier binding method in which an enzyme is physically or chemically bound to the surface of an insoluble carrier (see, for example, Patent Document 1), and a crosslinking agent having two or more functional groups. A comprehensive method that encapsulates the enzyme with a cross-linking method that forms an insoluble macromolecule by reacting the enzyme with the surface functional group of the enzyme, a lattice of a polymer gel having a network structure, or a water-insoluble microcapsule that includes an aqueous solution ( For example, refer to Patent Documents 2 to 4, and a composite method using these methods in combination (for example, refer to Patent Documents 5 and 6).

JP 59-213390 A JP 58-5193 A Japanese Patent Laid-Open No. 6-181763 Japanese Patent Publication No.53-12998 Japanese Patent Laid-Open No. 3-19687 Japanese Examined Patent Publication No. 62-16637

However, the carrier binding method has a large influence on the activity efficiency due to the size of the surface area due to the size of the carrier particles, and the processing method becomes a problem.
The cross-linking method requires a large amount of enzyme and has problems such as causing a decrease in enzyme activity upon reaction with the cross-linking agent.
In addition, the entrapment method has a problem that when a polymerization reaction is required for forming a matrix, the enzyme is easily deactivated during the polymerization.

The present invention has been made in view of such circumstances. An immobilized enzyme nanofiber that is less likely to inactivate an enzyme during production, has high enzyme activity, and is stable over a long period of time, a method for producing the same, and the nanofiber are used. An object of the present invention is to provide a reaction apparatus.

In the immobilized enzyme nanofiber according to the first invention that meets the above object, the enzyme is immobilized in a fibrous polymer matrix having a diameter of 100 nm to 3 μm that is spun by electrospinning.
The “electrospinning method” is a kind of spinning method that is also called an electrostatic spinning method, an electrospinning method, an electrification induced spinning method, or the like, and is a capillary-like shape to which a high voltage of several tens of kV is applied. Spinning is performed by extruding a solution containing a polymer compound at a constant speed from a spinning port (positive electrode) toward a grounded recovery plate (negative electrode).

In the immobilized enzyme nanofiber according to the first invention, the enzyme may be a lipase.

In the immobilized enzyme nanofiber according to the first invention, the polymer matrix may contain a siloxane polymer formed by polycondensation of an alkoxysilane compound.
In this case, the siloxane polymer is preferably a copolymer produced by polycondensation of a mixture of dialkyl dialkoxysilane and tetraalkoxysilane.
The “siloxane polymer” refers to linear, branched, and cyclic polymers represented by the general formula (SiR ¹ R ² O—) _n . R ¹ and R ² each independently represent a hydroxyl group, an alkyl group, or an aryl group, and n represents a natural number of 2 or more. For example, dialkylsiloxane is a homopolymer or copolymer containing siloxane units in which R ¹ and R ² are both alkyl groups.

The method for producing an immobilized enzyme nanofiber according to the second invention includes a step of preparing a raw material solution containing a spinnable polymer compound and an enzyme, and a step of spinning the raw material solution by an electrospinning method.

In the method for producing an immobilized enzyme nanofiber according to the second invention, the enzyme may be a lipase.

In the method for producing an immobilized enzyme nanofiber according to the second invention, the raw material solution further contains a sol of one kind or two or more kinds of alkoxysilane compounds, and a siloxane is obtained by polycondensation of an alkoxysilane compound in the spinning step. A polymer may be formed.
In this case, the alkoxysilane compound is preferably a mixture of dialkyl dialkoxysilane and tetraalkoxysilane.

The reaction apparatus according to the third invention uses a nonwoven fabric formed of the immobilized enzyme nanofibers according to the first invention as an immobilized enzyme carrier.

In the enzyme-immobilized nanofibers according to claims 1 to 4, compared with a conventional immobilized enzyme carrier such as a granular material, the surface area per unit volume is large, and more enzyme is exposed on the surface, The reaction efficiency is high because the molecular diffusion distance between the enzyme and the substrate can be shortened. Furthermore, it is possible to change the composition of the polymer matrix according to the enzyme to be immobilized and the characteristics of the enzyme reaction. It is also possible to use an enzyme whose surface has been chemically modified to improve its activity and stability. it can.

In particular, in the immobilized enzyme nanofiber according to claim 2, since lipase is used as an enzyme, such industries as hydrolysis of ester such as fatty acid triglyceride, ester synthesis, transesterification, synthesis of acid amide from amino acid (aminolysis), etc. It is applicable to useful reactions.

In the immobilized enzyme nanofiber according to claim 3, since the polymer matrix contains a siloxane polymer, the thermal stability of the enzyme immobilized in the matrix can be improved and the enzyme activity can be improved. it can.

In the immobilized enzyme nanofiber according to claim 4, since the siloxane polymer is a copolymer formed by polycondensation of a mixture of dialkyl dialkoxysilane and tetraalkoxysilane, the siloxane polymer is tertiary in the nanofiber. An original cross-linked structure is formed, the reaction substrate is easily accessible to the enzyme, and the enzyme activity can be improved.

In the method for producing an immobilized enzyme nanofiber according to claims 5 to 8, an immobilized enzyme nanofiber having a high reaction efficiency can be produced simply and at low cost. Moreover, since spinning can be performed under mild conditions, inactivation of the enzyme in the production process can be suppressed. Furthermore, since it can be applied to many enzymes and polymers, it is possible to optimize the composition of the polymer matrix according to the enzyme to be immobilized and the characteristics of the enzyme reaction.

In the method for producing an immobilized enzyme nanofiber according to claim 6, since lipase is used as an enzyme, industrial hydrolysis such as hydrolysis of ester such as fatty acid triglyceride, ester synthesis, transesterification, synthesis of acid amide from ester, etc. An immobilized enzyme nanofiber applicable to useful reactions can be provided.

In the method for producing an immobilized enzyme nanofiber according to claim 7, since a siloxane polymer is formed by polycondensation of an alkoxysilane compound in the spinning step, the thermal stability of the enzyme immobilized in the nanofiber is improved. And the enzyme activity can be improved.

In the method for producing an immobilized enzyme nanofiber according to claim 8, since the alkoxysilane compound is a mixture of a dialkyl dialkoxysilane and a tetraalkoxysilane, the siloxane polymer has a three-dimensional crosslinked structure in the nanofiber. This makes it easier to access the enzyme of the reaction substrate, and the enzyme activity can be improved.

In the reaction apparatus according to claim 9, since the nonwoven fabric formed by the immobilized enzyme nanofibers according to claims 1 to 4 is used as the immobilized enzyme carrier, the reaction efficiency is high and the enzyme can be used repeatedly. Therefore, the economic efficiency can be improved as compared with the batch method. In addition, since the immobilized enzyme carrier is a nonwoven fabric, it is easy to increase the amount of enzyme per unit volume while ensuring the circulation of the reaction liquid by laminating a plurality of sheets.

Next, embodiments of the present invention will be described with reference to the accompanying drawings for understanding of the present invention. Here, FIG. 1 is a schematic explanatory view of an electrospinning apparatus used for producing an immobilized enzyme nanofiber according to the first embodiment of the present invention.
In the immobilized enzyme nanofiber 14 (see FIG. 1) according to the first embodiment of the present invention, an enzyme is immobilized in a fibrous polymer matrix having a diameter of 100 nm to 3 μm. The immobilized enzyme nanofiber 14 uses a method having a step of preparing a raw material solution containing a spinnable polymer compound and an enzyme, and a step of spinning the raw material solution thus obtained by an electrospinning method. Manufactured.

As shown in FIG. 1, an electrospinning apparatus 10 used for manufacturing an immobilized enzyme nanofiber 14 is a syringe pump 12 for sending a raw material solution (hereinafter referred to as a solution) 11 as a raw material for spinning at a constant flow rate. And a capillary-shaped spinneret 13 for ejecting the solution 11 delivered from the syringe pump 12 and a recovery plate (collector) 15 for recovering the generated immobilized enzyme nanofibers 14. The spinneret 13 and the recovery plate 15 are arranged so that the interval is about several tens of centimeters, a positive voltage of several tens of kV is applied to the spinneret 13, and the recovery plate 15 is grounded. Therefore, the solution 11 is ejected from the spinneret 13 in a positively charged state. When the electrostatic attractive force is larger than the surface tension of the solution 11, a jet of the charged solution 11 is generated and flies toward the recovery plate 15. The solvent in the jet gradually evaporates, and the diameter of the jet decreases to the nanometer order when reaching the recovery plate 15. The immobilized enzyme nanofibers 14 thus formed are deposited on the recovery plate 15 and recovered as a mat-like nonwoven fabric. The immobilized enzyme nanofibers 14 can also be collected as a thread-like oriented body by rotating the collection plate 15 or providing a winding mechanism.

The diameter of the immobilized enzyme nanofiber is in the range of 100 nm to 3 μm. When the diameter is less than 100 nm, the mechanical strength is reduced and spherical defects called beads are easily generated. Moreover, since the ratio of a surface area will fall when a diameter exceeds 3 micrometers, enzyme activity falls.
The diameter of the immobilized enzyme nanofiber is determined by the concentration of the enzyme and polymer compound in the solution 11 as a raw material, the delivery speed from the syringe pump 12, the thickness of the spinneret 13, the potential of the spinneret 13, the spinneret 13 and the recovery. Although it can adjust by the space | interval with the board | substrate 15, etc., since the relationship between each of these factors and the diameter of the immobilized enzyme nanofiber 14 obtained depends on the kind of polymer compound, enzyme, and solvent to be used, it is unambiguous. It is difficult to make a decision. Therefore, it is preferable to examine in advance by a preliminary experiment or the like.

In the production of the immobilized enzyme nanofiber, the polymer compound that can be used as a material for the polymer matrix is not particularly limited, and any polymer compound that can be spun by an electrospinning method can be used.
Specific examples of the polymer compound that can be used include polyethylene, polystyrene, polyacrylonitrile, polyvinyl alcohol (PVA), poly L-lactic acid (PLLA), poly (lactic acid-glycolic acid copolymer) (PLGA), polyethylene oxide. Synthetic polymer compounds such as polyurethane, polyurea, polycarbonate, polyimide, polycaprolactone, polysaccharides such as chitin, chitosan, alginic acid, hyaluronic acid, dextran, collagen, gelatin, polyarginine, γ-polyglutamic acid (γ-PGA), Examples thereof include polypeptide polymer compounds such as silk, silica or siloxane polymers, inorganic polymer compounds such as alumina and titania, and mixtures thereof. Based on these, high molecular compounds (for example, vinylon) whose hydrophilicity, hydrophobicity, polarity and the like are changed by a technique such as chemical modification can also be used.

The polymer compound used as a material for the polymer matrix is appropriately selected according to the type of enzyme to be immobilized, the solvent used for the enzyme reaction, and the like. For example, when water is used as the reaction solvent, hydrophobic polymer compounds such as polystyrene and polycarbonate are used to prevent swelling and dissolution of the polymer matrix.
Conversely, when an organic solvent such as octane is used as the reaction solvent, hydrophilic polymer compounds such as PVA and polyethylene oxide are used in order to prevent swelling and dissolution of the polymer matrix. In the case of using a hydrophilic polymer compound, an effect of stabilizing the enzyme and suppressing deactivation by interaction with a hydrophilic group on the surface of the enzyme molecule can be expected.
Alternatively, in order to improve spinnability, a hydrophobic polymer compound and a hydrophilic polymer compound may be mixed and used, and after forming the immobilized enzyme nanofibers, one may be removed by washing with a solvent.

When the siloxane polymer is used alone or in combination with another polymer compound as the polymer compound, alkoxysilane which is a precursor of the siloxane polymer, water, and acid or ammonia as the hydrolysis catalyst in the solution 11 At the same time as the spinning step by electrospinning, a siloxane polymer is formed by, for example, hydrolysis of an alkoxysilane compound and polycondensation of the generated silanol (so-called sol-gel method) as shown in the following formula: You can also.

nSi (OR) ₄ +4 nH ₂ O → nSi (OH) ₄ +4 nROH
nSi (OH) ₄ → nSiO ₂ + 2nH ₂ O
In the above formula, n is an arbitrary natural number.

Examples of the alkoxysilane compound that can be used include a tetraalkoxysilane represented by the following general formula (1), a monoalkyltrialkoxysilane represented by (2), and a dialkyldialkoxysilane represented by (3). Is mentioned.
Si (OR) ₄ (1)
SiR ¹ (OR) ₃ (2)
SiR ¹ R ² (OR) ₂ (3)
In the formulas (1) to (3), R is a methyl (Me) group or an ethyl (Et) group, and R ¹ and R ² are each independently an alkyl group optionally having an arbitrary substituent. Or represents an aryl group.

Specific examples of the tetraalkoxysilane represented by the general formula (1) include tetramethoxysilane (TMOS, R = Me) and tetraethoxysilane (TEOS, R = Et).
Specific examples of the monoalkyltrialkoxysilane represented by the general formula (2) include methyltrimethoxysilane (MTrMOS, R = R ¹ = Me), methyltriethoxysilane (MTrEOS, R = Et, R ¹ = Me). ), 3-aminopropyltrimethoxysilane _{^{(APrTMOS, R = Me, R}} 1 = NH 2 CH 2 CH 2 CH 2 -), 3- aminopropyltriethoxysilane ^{(APrTrEOS, R = Et, R} 1 = NH 2 CH ₂ CH ₂ CH ₂ -), butyl trimetrexate Shishishi run _{^{(BuTrMOS, R = Me, R}} 1 = C 4 H 9), n- butyl triethoxysilane ^{(n-BuTrEOS, R = Et} , R 1 = C 4 H 9 ), phenyltrimethoxysilane _{^{(PheTrMOS, R = Me, R}} 1 = C 6 H 5), phenyl triethoxysilane Run _{^{(PheTrEOS, R = Et, R}} 1 = C 6 H 5), 3- trimethoxysilylpropyl methacrylate _{^{(TSM, R = Me, R}} 1 = CH 2 = C (CH 3) COO (CH 2) 3 -) Etc.
Specific examples of the dialkyl dialkoxysilane represented by the general formula (3) include dimethyldimethoxysilane (DMDMOS, R = R ¹ = R ² = Me), dimethyldiethoxysilane (DMDEOS, R = Et, R ¹ = R ² = Me), dibutyldimethoxysilane (DBDMOS, R = R ¹ = R ² = C ₄ H ₉ ), dibutyldiethoxysilane (DBDEOS, R = Et, R ¹ = R ² = C ₄ H ₉ ), etc. Can be mentioned.
These alkoxysilane compounds may be used alone or in combination of two or more (for example, a mixture of dialkyl dialkoxysilane and tetraalkoxysilane). A homopolymer and a copolymer (copolymer) are produced by polycondensation, respectively.

The types, combinations, concentrations and composition ratios of alkoxysilanes used in the production of immobilized enzyme nanofibers, and the concentration ratios with other polymer compounds depend on the type of enzyme used, the type of solvent used in the enzyme reaction, etc. Are appropriately selected.
For example, when an enzyme such as lipase whose reactivity is improved by a hydrophobic environment existing in the vicinity of the reaction center is used, the composition ratio of the alkoxysilane compound represented by the general formulas (2) and (3) is increased. It is preferable. Moreover, when the composition ratio of the alkoxysilane compound represented by the general formulas (1) and (2) is increased, crosslinking between siloxane chains can be formed, and the leakage of the enzyme can be suppressed while the enzyme is a reaction substrate. Access can be improved.

Any alkoxysilane compound other than the above can be used as long as it is an alkoxysilane compound that forms a siloxane polymer through hydrolysis and polycondensation reactions. Polysilica and sodium silicate can also be used.
Furthermore, an alkoxide of aluminum, zirconium, or titanium can be used in place of the alkoxysilane.

There is no restriction | limiting in particular in the enzyme which can be used for manufacture of an immobilized enzyme nanofiber, Arbitrary enzymes which are not inactivated by an electrospinning method can be used. In addition, for the production of immobilized enzyme nanofibers, an enzyme derived from a living body (so-called native enzyme) may be used as it is, and the protein structure is modified or modified for activity, thermal stability, and optimum pH modification. You may use the enzyme which gave. For the modification or modification of the enzyme, any of chemical methods, genetic engineering methods, and molecular evolution engineering methods may be used.

Examples of enzymes that can be used for the production of immobilized enzyme nanofibers include various enzymes shown below.
(1) Formate dehydrogenase, formaldehyde dehydrogenase, alcohol dehydrogenase, glyceraldehyde phosphate dehydrogenase, isocitrate dehydrogenase, malate dehydrogenase, glucose oxidase, cholesterol oxidase, L-amino acid oxidase, D-amino acid oxidase, lipoxygenase, urate oxidase, xanthine Oxidoreductases such as oxidase, protocatechuic acid-3,4-dioxygenase, pyridine nucleotide transhydrogenase, horseradish peroxidase (HRP), catalase, L-glutamate dehydrase.
(2) Lactate dehydrogenase (LDH), glutamate-pyruvate transaminase, creatine kinase, pyruvate kinase, hexokinase, thrombokinase, urokinase, streptokinase, carbamate kinase, transaldolase, phosphorylase, polynucleotide phosphorylase, dextransclase, tRNA- Transferases such as nucleotidyl transferase and NAD pyrophosphorylase.

(3) α-amylase, β-amylase, aldolase, invertase, diastase, β-glucosidase, β-fructofuranosidase, amyloglucosidase, lactase, glucoamylase, dextranase, takaamylase A, hyaluronidase, alkaline proteolytic enzyme , Neutral proteolytic enzyme, trypsin, α-chymotrypsin, σ-chymotrypsin, papain, subtilisin, subtilopeptidase A, subtilopeptidase B, pepsin, carboxypeptidase, renin, aminopeptidase M, leucine aminopeptidase, apyrase, Naringinase, kallikrein, elastase, chymase, ficin, pronase, asparakinase, aspartase, bromelin, rennin, prolidase, lipper Acetylcholinesterase, aminoacylase, steroid esterase, acid phosphatase, alkaline phosphatase, fructose diphosphatase, inorganic pyrophosphatase, aminoacylase, aminoacylase 1, ATPase, ATP deaminase, AMP deaminase, ribonuclease, ribonuclease T1, myosin ATPase, deoxy Hydrolytic enzymes such as ribonuclease, deoxyribonuclease 1, penicillin amidase, penicillinase, parathion hydrolase, atrazine chlorohydrolase, urease, lysozyme, thrombin, allylsulfatase, and D-oxynitrilase.

(4) Desorption enzymes such as phenylalanine decarboxylase and carbonic anhydrase.
(5) Isomerase such as α-amino-ε-caprolactam racemase, glucose isomerase, glucose phosphate isomerase.
(6) Synthetic enzymes (ligases) such as citrate synthase and succinyl CoA synthase.

In addition, modifications and modifications of these enzymes, mixtures thereof, or muscle enzyme extracts may be used. For example, functional proteins other than enzymes such as metmyoglobin and cytochrome C may be used.

The enzyme is appropriately selected according to the use of the immobilized enzyme nanofiber (type of enzyme reaction, substrate and product). For example, protein hydrolases such as trypsin and α-chymotrypsin are used for the production of amino acids by hydrolysis of proteins and peptides, and lipase is used for the production of fatty acids by hydrolysis of lipids and fatty acid esters. These hydrolases can also be used as synthesis catalysts for esters or acid amides, which are reverse reactions, by using an organic solvent as a reaction solvent.
Moreover, as uses other than production of useful substances, for example, application to biosensors such as quantification of glucose concentration in a solution using glucose oxidase can be mentioned.

The solution 11 used as a raw material for spinning by the electrospinning method is prepared by dissolving the polymer compound and the enzyme as described above in an appropriate solvent. As the solvent, a single solvent or a mixed solvent capable of dissolving both the polymer compound and the enzyme at an appropriate concentration is preferably used. For example, when the polymer compound is a water-soluble polymer compound such as PVA, water that is a good solvent for the enzyme is used.
The concentrations of the polymer compound and the enzyme contained in the solution 11 are appropriately adjusted according to the type and combination of the polymer compound and enzyme used, the diameter of the immobilized enzyme nanofiber 14 to be spun, and the like.

Next, a reaction apparatus according to the second embodiment of the present invention will be described. The reaction apparatus uses the immobilized enzyme nanofiber 14 of the above embodiment as an immobilized enzyme carrier. The reaction apparatus may be either a batch type (batch type) or a flow type, and the scale (internal volume, etc.) of the reaction apparatus is not particularly limited.
The immobilized enzyme carrier using the immobilized enzyme nanofibers 14 is preferably a non-woven fabric that does not hinder the flow of the reaction solution while ensuring a contact area with the reaction solution, and can be used by laminating a plurality of sheets. .

Next, examples carried out for confirming the effects of the present invention will be described. In the following examples, a case where lipase is used as an enzyme and polyvinyl alcohol is used as a polymer compound will be described, but the present invention is not limited to these combinations.

(1) Examination of PVA nanofiber production conditions by electrospinning method Using the electrospinning apparatus shown in FIG. 1, PVA aqueous solution not containing lipase was used as a raw material solution, and under various conditions (PVA aqueous solution concentration, applied voltage, Injection speed, distance between electrode plates (spinning port-collection plate distance)) PVA nanofibers were produced. The obtained nonwoven PVA nanofiber was observed with a scanning electron microscope, and the fiber diameter was measured.
As a result of examining the influence of the PVA aqueous solution concentration, applied voltage, injection speed, and electrode plate distance on the fiber diameter of the PVA nanofiber, the PVA aqueous solution concentration was 10% by weight, the applied voltage was 15 kV, the injection speed was 1.0 mL / hour, and It was found that nanofibers with a fiber diameter of about 0.75 μm can be obtained with good reproducibility when the distance between electrode plates is 10 cm. In the following examples, nanofibers were prepared under these conditions.

(2) Preparation of lipase-immobilized PVA nanofiber and evaluation of enzyme activity 180 mL of purified water and 20 g of PVA (Sigma-Aldrich, hydrolysis rate 98-99%, average molecular weight 146,000-186,000, the same applies hereinafter) Were mixed and dissolved by boiling in water for several hours. 15 mL of the 10% aqueous solution of PVA thus obtained was taken and 150 mg (1% by weight) of lipase (Wako Pure Chemical Industries, Ltd., lipase F-AP, the same applies hereinafter) was dissolved, and electrospinning (applied voltage 15 kV, injection speed 1. Non-woven lipase-immobilized PVA nanofibers were produced using 0 mL / hour and a distance between electrode plates of 10 cm).
The lipase-immobilized PVA nanofibers thus obtained were vacuum-dried, and the section cut out to a mass of 0.1 g was hydrated on a saturated LiCl aqueous solution at room temperature for 24 hours. The hydrated powder thus obtained was dispersed in 40 mL of an isooctane solution containing (S) -glycidol 20 mM and vinyl acetate 0.4 M as a substrate, and lipase was catalyzed under conditions of 35 ° C. and stirring speed 300 rpm. A transesterification reaction (see the following formula) was carried out.

Sampling was performed 4 times every 5 minutes from the start of the reaction, and the concentration of acetic acid (S) -glycidyl as a reaction product was monitored by gas chromatography (GLC). When the initial rate of reaction was determined from the change in acetic acid (S) -glycidyl concentration with time, a value of 6.3 μM / mg / min was obtained as the initial rate of reaction per 1 mg of lipase.
In addition, when the same measurement was performed, a value of 2.0 μM / mg / min was obtained as the initial reaction rate.
Moreover, when the same measurement was performed using the lipase inclusion PVA powder which freeze-dried and pulverized the solution used for the electrospinning method, the value of 4.8 micromol / mg / min was obtained as initial reaction rate. From this result, it can be seen that the enzyme activity is increased by using nanofibers.

(3) Preparation of lipase-immobilized PVA-polysiloxane nanofiber Any one of 0.136 mmol of TMOS (tetramethoxysilane), dimethyldimethoxysilane (DMDMOS), and a 4: 1 (molar ratio) mixture of DMDMOS-TMOS (0. 681 mmol) was mixed with 23.3 μL of purified water and 1.5 μL of 40 mM hydrochloric acid, respectively, for hydrolysis. While cooling this with ice water, 50 μL of 100 mM phosphate buffer (Na ₂ HPO ₄ -KH ₂ PO ₄ , pH 7.5) was mixed, and 150 mg of lipase dissolved in 167 μL of phosphate buffer was further added. The three types of solutions thus obtained were each added to 15 g of 10% PVA aqueous solution and stirred well, and then an electrospinning method (applied voltage: 15 kV, injection speed: 1.0 mL / hour, distance between electrode plates: 10 cm) was used. Thus, a non-woven lipase-immobilized PVA-polysiloxane nanofiber was produced.
Treated in the same manner as the lipase-immobilized PVA nanofiber prepared in (2), the obtained hydrated powder was dispersed in 40 mL of an isooctane solution containing (S) -glycidol 20 mM and vinyl acetate 0.4 M as a substrate, The transesterification was performed under conditions of 35 ° C. and a stirring speed of 300 rpm. Sampling was performed 4 times every 5 minutes from the start of the reaction, and the concentration of acetic acid (S) -glycidyl as a reaction product was monitored by gas chromatography (GLC). The initial rate of reaction was determined from the time change of the acetic acid (S) -glycidyl concentration.
The obtained results are shown in Table 1 together with the results obtained in (2).

In Table 1, no. 1 and no. 2 represents a powdered lipase dispersed directly in an isooctane solution of a reaction substrate and a hydrated powder obtained from a lipase-immobilized PVA nanofiber (see (2) above). 3 to 6 represent hydrated powders obtained from lipase-immobilized PVA-polysiloxane nanofibers prepared under various conditions in this example.
No. From comparison between 1 and 2, it can be seen that the enzyme activity is improved by immobilizing lipase in PVA.
No. 2 and No. From the comparison with the results of 3 and 4, the introduction of the TMOS-derived siloxane polymer into the polymer matrix increases the hydrophilicity in the vicinity of the lipase, but the enzyme activity rather decreases, but the polymer of the DMDMOS-derived siloxane polymer. It can be seen that introduction into the matrix improves the enzyme activity by providing a hydrophobic environment near the lipase.

In addition, No. 5 is a result obtained from a lipase-immobilized nanofiber prepared from a solution obtained by freeze-drying a mixed solution of DMDMOS hydrolyzate and lipase and adding the obtained freeze-dried powder to a 10% PVA aqueous solution. Although the enzyme activity is slightly reduced as compared with the case where lyophilization was not performed (No. 4), It can be seen that the enzyme activity is higher than those of 1 and 2.
No. From comparison between 4 and 6, it can be seen that introduction of DMDMOS and TMOS into the polymer matrix further improves the enzyme activity. This is considered to be due to the fact that the reaction substrate easily comes into contact with the lipase because a three-dimensional skeleton was formed by the formation of a bridge between siloxane chains.

(4) Covering the lipase-immobilized PVA nanofibers with the siloxane gel (2) After drying the non-woven lipase-immobilized PVA nanofibers in a vacuum and cutting them out so that the mass becomes 0.1 g, The sample was immersed in a 5% DMDMOS hexane solution heated for 5 hours with stirring. The lipase-immobilized PVA nanofibers taken out from the solution were coated with a siloxane gel produced from DMDMOS hydrolyzed by moisture in the air. When the enzyme activity was measured by the above-mentioned method after vacuum drying, a value of 12 μM / mg / min was obtained as the initial reaction rate. The results are shown in Table 1. Compared with the result of 2, it can be seen that the enzyme activity is improved as compared with the case where only PVA is used as the polymer matrix. From this, it was confirmed that the enzyme activity of the lipase can also be improved by immobilizing the lipase in the PVA matrix and then treating it with DMDMOS.

(5) Preparation of reactor using lipase-immobilized PVA nanofibers and lipase-immobilized PVA-polysiloxane nanofibers and performance evaluation The same method as in (2), vacuum drying, and hydrated nonwoven fabric A lipase-immobilized PVA nanofiber, and a non-woven lipase-immobilized PVA-polysiloxane nanofiber prepared in the same manner as in (3) (using a DMDDOS-TMOS 4: 1 mixture), vacuum-dried, and hydrated into a lipase-immobilized PVA-polysiloxane nanofiber Each was cut with a 6 mm diameter punch and charged into a cylindrical flow reactor.
An isooctane solution containing (S) -glycidol 20 mM and vinyl acetate 0.4 M as a substrate is passed at a constant flow rate (1.0, 0.5, 0.1 mL / min) and sampled every 10 minutes from the start of the reaction. And the conversion rate to the reaction product, acetic acid (S) -glycidyl, was monitored by gas chromatography (GLC). The value at the time when the conversion rate became constant was taken as the steady reaction rate. The results are as shown in Table 2.

It can be seen that the smaller the flow rate, the higher the steady-state reaction rate, and the lipase-immobilized PVA-polysiloxane nanofibers show a higher steady-state reaction rate than the lipase-immobilized PVA nanofibers.

As a result of carrying out the same measurement as described above at a flow rate of 0.5 mL / min, with 27 and 100 lipase-immobilized PVA nanofibers and lipase-immobilized PVA-polysiloxane nanofibers, respectively, charged in the reactor. Is shown in Table 3.

It can be seen that, in both cases of lipase-immobilized PVA nanofibers and lipase-immobilized PVA-polysiloxane nanofibers, the steady-state reaction rate is improved by increasing the number of laminated layers.

It is a schematic explanatory drawing of the electrospinning apparatus used for manufacture of the immobilized enzyme nanofiber which concerns on the 1st Embodiment of this invention.

Explanation of symbols

10: Electrospinning device, 11: Solution, 12: Syringe pump, 13: Spinning port, 14: Immobilized enzyme nanofiber, 15: Recovery plate

Claims (9)

An immobilized enzyme nanofiber, wherein an enzyme is immobilized in a fibrous polymer matrix having a diameter of 100 nm to 3 μm spun by electrospinning. The immobilized enzyme nanofiber according to claim 1, wherein the enzyme is a lipase. The immobilized enzyme nanofiber according to any one of claims 1 and 2, wherein the polymer matrix contains a siloxane polymer formed by polycondensation of an alkoxysilane compound. Enzyme nanofiber. The immobilized enzyme nanofiber according to claim 3, wherein the siloxane polymer is a copolymer formed by polycondensation of a mixture of a dialkyl dialkoxysilane and a tetraalkoxysilane. . A method for producing an immobilized enzyme nanofiber, comprising a step of preparing a raw material solution containing a spinnable polymer compound and an enzyme, and a step of spinning the raw material solution by an electrospinning method. The method for producing an immobilized enzyme nanofiber according to claim 5, wherein the enzyme is a lipase. The method for producing an immobilized enzyme nanofiber according to any one of claims 5 and 6, wherein the raw material solution further contains a sol of one or two or more types of alkoxysilane compounds, and the alkoxy in the spinning step. A method for producing an immobilized enzyme nanofiber, wherein a siloxane polymer is produced by polycondensation of a silane compound. The method for producing an immobilized enzyme nanofiber according to claim 7, wherein the alkoxysilane compound is a mixture of a dialkyl dialkoxysilane and a tetraalkoxysilane. A reaction apparatus using the nonwoven fabric formed of the immobilized enzyme nanofiber according to any one of claims 1 to 4 as an immobilized enzyme carrier.