JP3543994B2 - Enzyme immobilization method and immobilized enzyme - Google Patents

Description

【００７４】
【発明の効果】
上記したように本発明は、非水系溶媒中でも高い触媒作用を発現する。又、容易に回収、再利用し得る。
【図面の簡単な説明】
【図１】Ｏ／Ｗエマルションの模式図。
【図２】Ｏ／Ｗ／Ｏエマルションの模式図。
【図３】Ｏ／Ｗ／Ｏエマルションの模式図。
【図４】ＰＥＧの分子量の影響。
【符号の説明】
１ 被覆酵素
２ ポリマー
３ 水溶液
４ ポリマー溶液
５ 有機溶媒
６ Ｏ／Ｗエマルション
７ 内有機相
８ 水溶性ポリマーを含む水相
９ 界面活性剤被覆酵素
１０ 外有機相
１１ 界面活性剤[0001]
[Industrial applications]
The present invention relates to a method for preparing an immobilized enzyme which exhibits a high catalytic activity even in a non-aqueous solvent by a very simple method and can be easily recovered and reused. The present invention brings great progress in the production of organic chemicals useful in the industrial, agricultural and marine industries, cosmetics, and organic chemical industries, for example, alcohols, carboxylic acids, esters, and peptides.
[0002]
[Prior art]
Attempts to produce organic compounds such as physiologically active substances and chemical products by applying the reaction specificity of enzymes have been made for a long time.
[0003]
However, enzymes are generally soluble in aqueous solutions and can be stably present only in aqueous solutions whose pH has been adjusted.
[0004]
Therefore, in order to apply the catalysis of the enzyme to organic synthesis, the reaction is preferably performed in an aqueous solution, and if performed in a non-aqueous solvent, the higher order of the enzyme itself is generated by the interaction between the solvent and the enzyme. In most cases, the structure is broken and the ability as a catalyst is rapidly lost (deactivation).
[0005]
When applying the catalytic action of enzymes to organic synthesis reactions, there is relatively little problem if the substrate and reactants are water-soluble.
[0006]
However, in organic synthesis, in most cases, the substrate is dissolved only in a non-aqueous solvent and can be reacted only in a non-aqueous solvent.If the enzyme is used as it is in a non-aqueous solvent, Very expensive enzymes are almost immediately deactivated or show no catalysis.
[0007]
Attempts to overcome the above problems and utilize enzymes in non-aqueous solvents have been made for some time, and as such means, enzyme modification, immobilization on carriers and the like are known.
[0008]
However, these techniques are difficult because they require very complicated and sophisticated technology and are not generally applicable to any enzyme.
[0009]
Even if the enzyme can be modified to be stably present in a non-aqueous solvent by these methods, there is a very high possibility that the intrinsic activity of the enzyme will be significantly reduced.
[0010]
As one of attempts to use an enzyme in a non-aqueous solvent, an invention using an enzyme whose surface is protected by a lipid molecule is described in JP-A-64-80282.
[0011]
According to the invention, a powdery enzyme-lipid complex is formed by dissolving an enzyme in an aqueous pH buffer solution and directly dispersing lipids therein.
[0012]
The composite powder is easily soluble in a non-aqueous solvent and exhibits a reaction catalysis specific to an enzyme in the non-aqueous solvent.
[0013]
However, the complex is easily soluble in the non-aqueous solvent, and it is difficult to recover the complex after the completion of the catalytic reaction because of the high solubility.
[0014]
Generally, an organic synthesis reaction utilizing the catalytic action of an enzyme has high selectivity and has few by-products. Therefore, if the used enzyme can be efficiently recovered from the reaction system by a simple method, it is easy to separate the product from the reaction solution.
[0015]
However, the only way to remove the enzyme dissolved in the non-aqueous solvent is to separate it after re-precipitating the enzyme using the solubility or to use a separation column.
[0016]
However, usually, the precipitation method has a large loss only by utilizing the difference in solubility due to temperature change, and the column requires labor and cost, and in some cases, the activity is significantly reduced during the separation operation. I will.
[0017]
Thus, although the enzyme-lipid complex is characterized in that the enzyme can be used in a non-aqueous solvent in a simpler manner, it is still unsatisfactory in reusing extremely expensive enzymes. It is something.
[0018]
[Problems to be solved by the invention]
The present inventors have conducted intensive studies to find a method for immobilizing an enzyme that shows a catalytic action even in a non-aqueous solvent, can be easily recovered after the reaction, and is easy to prepare. Have found a new invention that overcomes the problems of the present invention.
[0019]
[Means for Solving the Problems]
That is, the present invention relates to an immobilized enzyme which has an activity even in a non-aqueous solvent and can be recovered and reused, wherein the enzyme is coated and immobilized with a water-soluble polymer.
[0020]
The immobilized enzyme according to the present invention is in the form of powder and exhibits a high catalytic action even in a non-aqueous solvent.
[0021]
Since it is in a powder form and insoluble in a non-aqueous solvent, it is very easy to recover the enzyme after the reaction, it is almost quantitatively recovered, and it can be reused repeatedly.
[0022]
Further, the present invention provides a method for preparing an O / W emulsion using a non-hydrophilic organic solvent containing a surfactant-coated enzyme and an aqueous solution of a water-soluble polymer, and then preparing the O / W emulsion using the O / W emulsion and the non-hydrophilic organic solvent. The present invention relates to a method for preparing an immobilized enzyme having activity even in a non-aqueous solvent, characterized in that a surfactant-coated enzyme is included in water-soluble polymer particles by drying an / O emulsion.
[0023]
Surfactant coated enzyme used in the present invention is an enzyme the enzyme molecular surface is protected by interfacial active agent molecules, for example, be prepared by the method shown in JP 64-80282.
[0024]
That is, the enzyme is dissolved in an aqueous pH buffer solution, and the surfactant is directly dispersed therein to form a powdery surfactant-coated enzyme in which the surface of the enzyme molecule is protected by the surfactant molecule. It is prepared by drying.
[0025]
As an enzyme that can be used in the present invention, first, a hydrolase can be mentioned. As esterases that are hydrolytic enzymes, lipases derived from animals and microorganisms are used, and peptidases and amidases that hydrolyze peptides and amide groups include pepsin, α-chymotrypsin, thermolysin, promerin, aminopeptidase, arginase, urease, and penicillinase. It is mentioned as a typical thing.
[0026]
In addition, examples of glucosidase acting on sugar include oligosaccharides such as α- and β-glucosidase, α- and β-amylase, and polysaccharides.
[0027]
Other examples include isomerases such as stereoisomerase and structural isomerase, transferases such as transaminase and transglucosidase, oxidoreductases, and dehydrogenases.
[0028]
The surfactant used in the present invention includes all compounds having a structure in which a hydrophobic group represented by an alkyl group and the like and a hydrophilic group represented by various polar groups coexist in the molecule.
[0029]
Specifically, nonionic surfactants having a hydroxyl group or an ethylene glycol skeleton such as sorbitan alkyl ester, polyoxyethylene glycol alkyl ester, polyoxyglycol alkyl ether, and dialkyl glutamate gluconamide, and naturally occurring lipids, For example, glyceroglycolipid, glycosphingolipid, steroid glycoside and the like can be used.
[0030]
In consideration of the electrostatic properties of the enzyme used and the stability of the enzyme, cationic surfactants such as alkyl and dialkyl ammonium salts, fatty acids, alkyl sulfonic acids, dialkyl sulfosuccinic acids, dialkyl phosphates, etc. And an amphoteric surfactant such as sulfatidylcholine and phosphatidylserine can be used alone or as a mixture with the above-mentioned nonionic surfactant.
[0031]
As a specific method for obtaining a surfactant-coated enzyme, the above-mentioned surfactant is added alone or mixed to a buffer solution in which the enzyme is dissolved at a concentration of 0.1 g to 50 g / l so as to have a concentration of 1 to 500 g / l. Then, a precipitate generated by performing stirring or ultrasonic irradiation can be obtained by filtering and drying.
[0032]
It can also be obtained by forming a W / O emulsion with a non-hydrophilic organic solvent containing a surfactant and a pH buffer aqueous solution in which an enzyme is dissolved, and drying the W / O emulsion.
[0033]
Alternatively, a pH buffer aqueous solution in which an enzyme is dissolved is contacted with a non-hydrophilic solution in which the above-mentioned surfactant is dissolved, and the enzyme in the aqueous solution is reverse-phased by the surfactant in the non-hydrophilic solution phase. There is also a method of adjusting by incorporating in a micelle, and it is also possible to use this organic phase after drying in the next O / W emulsion adjustment process, or to use the organic phase as it is for the next O / W emulsion adjustment. .
[0034]
However, the method for preparing the surfactant-coated enzyme is not limited to these methods, but the weight ratio of the enzyme of the surfactant-coated enzyme used for immobilization to the surfactant is determined by the type of the enzyme. , And the type of surfactant used, and is usually in the range of 1: 3 to 1: 100.
[0035]
The water-soluble polymer was dissolved to about 5 to 50% in 10 parts of a solution in which the surfactant-coated enzyme thus obtained was dissolved in a non-hydrophilic organic solvent to a concentration of 0.1 to 50 g / l. The O / W emulsion is adjusted by mixing and stirring 5 to 100 parts of the aqueous solution and, if necessary, irradiating ultrasonic waves (FIG. 1).
[0036]
Non-hydrophilic organic solvents used for preparing the O / W emulsion include hydrocarbon solvents such as hexane, heptane and isooctane, aromatic hydrocarbon solvents such as benzene, toluene and xylene, and methyl isobutyl ketone. Such non-hydrophilic ketones, ester solvents such as ethyl acetate, and halogen solvents such as dichloromethane and chloroform can be used.
[0037]
Examples of the water-soluble polymer include polyethylene glycol, ethylene oxide-propylene oxide copolymer, polyacrylamide, polyacrylic acid or a salt thereof, acrylamide-acrylic acid or a salt thereof, a copolymer of tritylaminoethyl (meth) acrylate and a copolymer with acrylamide, Representative examples include polyvinylamine and salts thereof, polysaccharides, and CMC polymers.
[0038]
Although the molecular weight of the water-soluble polymer can be used as long as it is 1,000 or more, it is preferable to use one having a molecular weight of 5,000 or more in consideration of handling of surfaces such as stickiness when used as an immobilized enzyme. The molecular weight may be any high as long as it is water-soluble.
[0039]
When adjusting the O / W emulsion, if necessary, the above-mentioned surfactant may be added in the range of 0.1 to 10% by weight based on the weight of the non-hydrophilic organic solvent.
[0040]
10 parts of the obtained O / W emulsion and 5 to 300 parts of a non-hydrophilic organic solvent in which the above-mentioned surfactant is dissolved at a concentration of 0.1 to 20% by weight are mixed under stirring. The O / W / O emulsion is adjusted by mixing by sonic irradiation. As a result, a non-hydrophilic organic solvent containing a surfactant-coated enzyme is present in the innermost phase of the emulsion, a water-soluble polymer aqueous solution is present on the outside, and a non-hydrophilic organic solvent is present on the outside as a continuous phase. (FIGS. 2 and 3).
[0041]
The O / W / O emulsion thus obtained is naturally dried, dried under reduced pressure, or freeze-dried to prepare an immobilized enzyme in which a surfactant-coated enzyme is included in a porous powdery water-soluble polymer. Is done.
[0042]
Further, the present invention provides an immobilization method having a composition in which the weight ratio of polyethylene glycol to the enzyme is 100,000: 1 to 100: 1, and which is active even in a non-aqueous solvent. It is about enzymes.
[0043]
The immobilized enzyme obtained by the method according to the present invention, in which polyethylene glycol is used as the water-soluble polymer, has a weight ratio of polyethylene glycol to the enzyme itself of 100,000: 1 to 100: 1. When the ratio exceeds 100,000, the catalytic activity of the enzyme is low. When the ratio is less than 100, the enzyme immobilization efficiency is poor. , Leakage of the enzyme itself is increased, and the recovery rate is significantly reduced.
[0044]
The immobilized enzyme according to the present invention is obtained by physically enclosing a surfactant-coated enzyme in porous water-soluble polymer particles obtained by applying an O / W / O emulsion, Unlike the enzyme modification method and the immobilization method using the enzyme, the catalytic activity of the enzyme itself is not impaired.
[0045]
Further, in an attempt, the catalytic activity of a powder obtained by drying the O / W emulsion without using the O / W / O emulsion was examined, but no activity was observed.
[0046]
This may be because the O / W emulsion is destroyed during drying and is not included in the polyethylene glycol particles, or even if it is included, the porosity of the polyethylene glycol particles is poor, so that the contact between the substrate and the enzyme is extremely high. It seems that there are few.
[0047]
From this point as well, it was confirmed that in the method of the present invention, drying after forming a W / O / W emulsion is essential.
[0048]
The immobilized enzyme according to the present invention exhibits a catalytic action in a non-aqueous organic solvent, but as the non-aqueous solvent, a non-hydrophilic organic solvent is preferable, and a hydrocarbon solvent such as hexane, heptane, and isooctane, benzene, and toluene. , aromatic hydrocarbons such as xylene, and non-hydrophilic systems ketones such as methyl isobutyl ketone, ester such as ethyl acetate, dichloromethane, it is possible to use halogen-based solvent, such as chloroform.
[0049]
If necessary, a small amount of a hydrophilic organic solvent such as methanol, ethanol or acetone may be added.
[0050]
When the immobilized enzyme of the present invention is used, for example, as a catalyst for esterification, it is used as follows.
[0051]
That is, alcohol and carboxylic acid, which are raw materials for esterification, are dissolved in a non-aqueous solvent, and the powdery immobilized enzyme according to the present invention is added thereto, followed by keeping the temperature and stirring.
[0052]
After completion of the reaction, it can be easily recovered from the reaction solution by decantation or filtration. The recovered immobilized enzyme can be used repeatedly as many times as necessary.
[0053]
In addition, instead of the batch method as described above, for example, a tubular reactor is filled with the immobilized enzyme according to the present invention and reacted by passing a non-aqueous solvent in which the raw material is dissolved. Is also applicable.
[0054]
The immobilized enzyme and the enzyme immobilization method according to the present invention provide a great advance in the fields of medicine, agricultural chemicals, food chemistry, synthesis of clinical drugs, and analytical instruments utilizing an enzyme reaction.
[0055]
Embodiment 1
Pseudomonas sp. 3 g of the original lipase was dissolved in 500 ml of a phosphate buffer having a pH of 7, and 12.39 g of 2C18Δ9GE (formula 1) was dissolved therein, followed by irradiation with ultrasonic waves for 20 minutes.
[0056]
Then, the powder precipitated in the aqueous solution was separated by a centrifugal separator and dried under vacuum. As a result, 2.78 g of a surfactant-coated lipase as a white powder having a water content of 0.79% was obtained.
[0057]
The weight percentage of lipase in the complex was determined to be 20.6% by UV spectrum.
[0058]
The obtained surfactant-coated enzyme was dissolved in toluene at a concentration of 1.0 g / l. Polyethylene glycol having a molecular weight of 20,000 was dissolved in a buffer solution of pH 7 at a concentration of 10%.
[0059]
An O / W emulsion was prepared by mixing 3 ml of the above toluene solution and 15 ml of the above aqueous solution of polyethylene glycol, and stirring and mixing at 6000 rpm for 2 minutes using a homogenizer.
[0060]
Subsequently, the O / W emulsion was mixed with 100 ml of a toluene solution in which 2C18Δ9GE was dissolved at a concentration of 10 mol / m ³ , and the mixture was similarly stirred at 6000 rpm for 2 minutes to prepare an O / W / O emulsion. Then, the prepared O / W / O emulsion was concentrated and dried by an evaporator to obtain an enzyme immobilized on polyethylene glycol powder according to the present invention.
[0061]
Embodiment 2
And isooctane solution 5ml of lauric acid dissolved in a concentration of 6 mol / m ^3, was mixed isooctane solution 5ml of benzyl alcohol at a concentration of 6 mol / m ^3, the immobilized enzyme 0.42g created in Example 1 thereto After addition (substantially the concentration of the enzyme in the reaction solution was 0.054 g / l) and the mixture was kept at 30 ° C. for 1 hour, the product benzyl laurate was quantified by gas chromatography.
[0062]
After the reaction, 99% or more of the immobilized enzyme could be recovered by filtration. The recovered immobilized enzyme was washed with isooctane and used repeatedly for the reaction.
[0063]
The ester production yield (X) at the first hour of the start in each reaction is as shown in FIG. 4, and the recovery rate of the immobilized enzyme was 99% or more even when used three times repeatedly, and was almost quantitative. And no decrease in enzyme activity was observed.
[0064]
Embodiment 3
Using polyethylene glycol having a molecular weight of 1,000, 2,000, or 6,000, an immobilized enzyme was prepared according to the method described in Example 1, and each immobilized enzyme was used in the reaction according to the method described in Example 2. .
[0065]
As a result, as shown in FIG. 4, even if the immobilized enzyme using polyethylene glycol of each molecular weight was repeatedly used, the recovery of the immobilized enzyme was performed quantitatively simply by filtration, and almost no decrease in the activity was observed. I couldn't see it.
[0066]
[Comparative Example 1]
And isooctane solution 50ml of lauric acid dissolved in a concentration of 6 mol / m ^3, was mixed isooctane solution 50ml of base down benzyl alcohol was dissolved at a concentration of 6 mol / m ^3, the surfactant coating prepared therewith in Example 1 After adding 27 mg of lipase (concentration of the actual enzyme in the reaction solution: 0.054 g / l) and keeping the mixture at 30 ° C. for 1 hour, the product benzyl laurate was quantified by gas chromatography.
[0067]
As a result, formation of benzyl laurate was observed at a yield of 70%.
[0068]
After leaving the reaction solution at 0 ° C. for 2 days, the precipitate was separated. As a result, 10 mg of surfactant-coated lipase was recovered. The recovery was 37%.
[0069]
[Comparative Example 2]
The surfactant-coated enzyme prepared in Example 1 was dissolved in toluene at a concentration of 1.0 g / l. Polyethylene glycol having a molecular weight of 20,000 was dissolved in a buffer solution of pH 7 at a concentration of 10%.
[0070]
An O / W emulsion was prepared by mixing 3 ml of the above toluene solution and 15 ml of the above aqueous solution of polyethylene glycol, and stirring and mixing at 6000 rpm for 2 minutes using a homogenizer.
[0071]
Subsequently, the O / W emulsion was dried as it was without being changed to the O / W / O emulsion as in Example 1. The dried powder was used for the reaction as in Example 2.
[0072]
As a result, it was confirmed that the present powder did not show catalytic activity, and that it was indispensable to form an O / W / O emulsion as in the present invention in order to show the catalytic activity of the enzyme.
[0073]
Embedded image

[0074]
【The invention's effect】
As described above, the present invention exhibits a high catalytic action even in a non-aqueous solvent. Also, it can be easily collected and reused.
[Brief description of the drawings]
FIG. 1 is a schematic diagram of an O / W emulsion.
FIG. 2 is a schematic diagram of an O / W / O emulsion.
FIG. 3 is a schematic diagram of an O / W / O emulsion.
FIG. 4. Effect of PEG molecular weight.
[Explanation of symbols]
REFERENCE SIGNS LIST 1 coated enzyme 2 polymer 3 aqueous solution 4 polymer solution 5 organic solvent 6 O / W emulsion 7 inner organic phase 8 aqueous phase containing water-soluble polymer 9 surfactant-coated enzyme 10 outer organic phase 11 surfactant

Claims (2)

After adjusting the O / W emulsion with a non-hydrophilic organic solvent containing a surfactant-coated enzyme and an aqueous solution of polyethylene glycol, the O / W / O emulsion obtained from the O / W emulsion and the non-hydrophilic organic solvent is dried. it allows, characterized in that to encompass surfactant coated enzyme in a water-soluble polymer particles, immobilized enzyme preparation manufactured by a method having the activity in a non-aqueous solvent. Obtained by the method shown in claim 1, the weight ratio of polyethylene glycol and the enzyme of 100,000: 1 to 100: 1 and comprising a composition, immobilized enzymes have activity in a non-aqueous solvent.

Images