JP2007006717A - Method for producing immobilized enzyme - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for immobilizing an enzyme in an enzyme solution on a carrier with high efficiency according to a physical adsorption method using the enzyme solution containing a contaminant such as a microorganism cultured product. <P>SOLUTION: The method for producing an immobilized enzyme comprises carrying out an enzyme immobilizing step of bringing the enzyme solution containing the contaminant into contact with a carrier for immobilizing the enzyme according to the physical adsorption a plurality of times and thereby immobilizing the enzyme in the enzyme solution on the carrier. The method for producing the immobilized enzyme has a carrier washing step of washing the carrier at least once with a water-soluble organic solvent or an aqueous solution thereof during the enzyme immobilizing step at a plurality of times. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing an immobilized enzyme in which an enzyme in an enzyme solution containing impurities such as a microorganism culture is immobilized on a carrier.

Since the reaction using the enzyme which is a biocatalyst is a reaction using the high substrate specificity of the enzyme, the target product can be produced efficiently, which is advantageous for cost reduction. In addition, since the reaction is performed under mild conditions, it is an excellent method such that less energy is consumed and the environmental load can be reduced.
Therefore, the use of enzyme reaction has been studied industrially in many fields, but it is difficult to separate the enzyme and the reaction product by the method in which the enzyme and substrate are stirred and reacted in a solution. Since the enzyme cannot be efficiently recovered, it is economically difficult to industrialize the enzyme reaction particularly when an expensive enzyme is used.

  Therefore, a method for producing an immobilized enzyme by a carrier binding method in which an enzyme is immobilized on an insoluble carrier by covalent bond, ionic bond, physical adsorption, biochemical affinity, etc. has been studied for many years.

In particular, immobilization of an enzyme by physical adsorption can be immobilized on a carrier without modifying the enzyme, so the production process is simple and low-cost in the production of the immobilized enzyme, and the enzyme is deactivated. Is less likely to do. Therefore, it has a big advantage for industrial use (for example, refer nonpatent literature 1).

As an enzyme immobilization method by physical adsorption, for example, a protein solution containing an organic solvent is immobilized on a carrier, and the immobilized carrier does not contain an organic solvent or does not denature proteins. A method of washing with a buffer solution containing an organic solvent is known (see Patent Document 1). However, the immobilization method, for example, depending on the type of enzyme, such as a hydrophobic enzyme such as cutinase, when the organic solvent is contained in the solvent in the immobilization step, the enzyme remains dissolved, and the enzyme In some cases, the amount of immobilization dropped.
The world of bioreactors-its basics and applications for practitioners, pp25-pp61, 1992 Japanese Patent Laid-Open No. 11-174055

In addition, enzyme immobilization by physical adsorption force generally has a weak bond between the enzyme and the carrier, so it is necessary to consider the effects of immobilization temperature and coexisting substances during immobilization in the production of the immobilized enzyme. There is.
In particular, microbial cultures and the like contain many microorganism-derived contaminants such as various proteins or polysaccharides, and when such cultures are used as enzyme samples for immobilization, fixation with the contained contaminants is required. The amount of enzyme immobilized further decreases due to the inhibition of crystallization. For this reason, the enzyme sample to be used must be as pure as possible. However, the production of such a high-purity enzyme requires complicated and many purification steps, and is also costly. There was a problem that it was disadvantageous.

  Accordingly, an object of the present invention is to provide a method for immobilizing an enzyme in an enzyme solution on a carrier with high efficiency by a physical adsorption method using an enzyme solution containing impurities such as a microorganism culture. is there.

  As a result of intensive studies, the present inventors contacted an enzyme solution containing impurities and an immobilization carrier that can immobilize the enzyme by a physical adsorption method, and brought them into contact with each other in the enzyme solution. In the process of immobilizing an enzyme on a carrier, the amount of the enzyme immobilized on the carrier can be improved by washing the immobilized carrier with a water-soluble organic solvent or an aqueous solution thereof, thereby completing the present invention. It came.

  That is, the present invention immobilizes an enzyme in an enzyme solution on a carrier by performing an enzyme immobilization step in which an enzyme solution containing impurities and a carrier on which the enzyme is immobilized by physical adsorption are contacted multiple times. A method for producing an immobilized enzyme, comprising: a carrier washing step of washing the carrier at least once with a water-soluble organic solvent or an aqueous solution thereof between a plurality of enzyme immobilization steps. It is a manufacturing method of an enzyme.

  According to the present invention, it is possible to provide an immobilized enzyme in which an enzyme solution containing impurities such as a microorganism culture is immobilized on a carrier with high efficiency.

The present invention will be described in detail below.
(enzyme)
In the present invention, the enzyme used for immobilization on the carrier is not particularly limited, but preferred examples include lipase, esterase and cutinase. Of these, lipase and cutinase are particularly preferred.
Usually, an enzyme sample can be obtained by culturing a microorganism that produces it. However, in microbial culture, sugars and other proteins are simultaneously produced as impurities in addition to the target enzyme. These contaminants are produced intracellularly when the microorganism grows and then secreted outside the cell. Examples of such contaminants include monosaccharides such as glucose and galactose in the case of sugar. Examples of other proteins include various enzymes necessary for the growth of microorganisms. In addition, the sugars are complexed with lipids and proteins. There are many things such as those forming a body.

As an enzyme solution containing a large amount of impurities used in the present invention, for example, Cryptococcus sp S-2 (FERM P-15155, hereinafter abbreviated as Cryptococcus sp S-2), which is a kind of yeast, is used. A culture solution containing the cutinase to be produced can be mentioned as a preferable one.
Cutinase can be obtained, for example, by the following method. That is, after culturing Cryptococcus sp-2, which is a kind of yeast that produces cutinase, under culture conditions that are optimal for growth according to conventional methods, the cells are separated from the culture solution by centrifugation, and cutinase is obtained. A culture supernatant containing is obtained. The supernatant is filtered using, for example, an ultrafiltration membrane to obtain a cutinase enzyme solution. This cutinase enzyme solution is preferably a cutinase enzyme dry powder obtained by freeze-drying to maintain the enzyme activity for a long period of time, but the above operation for obtaining the dry powder is omitted, and the microorganism culture is performed. The solution can be used for immobilization on a carrier as it is.
Usually, when an enzyme is immobilized on a carrier, it is necessary to use a high-purity enzyme sample that has undergone many purification steps in order to increase the immobilization efficiency, but these microorganism culture solutions containing various contaminants Alternatively, the present invention is particularly effective for freeze-dried powders and the like, and the target enzyme can be immobilized on a carrier with high efficiency without performing the above complicated and many purifications.

(Carrier)
The enzyme immobilization carrier used in the present invention is a carrier capable of immobilizing an enzyme by a physical adsorption method, and the material may be either organic or inorganic, and the activity of the enzyme immobilized on the carrier is not lost and is handled. In addition, there is no particular limitation as long as it is physically and chemically stable.
Examples of the inorganic immobilization carrier include celite, diatomaceous earth, kaolinite, molecular sieve, porous glass, ceramic and the like. Examples of the organic immobilization carrier include acrylic resin and polyester resin. , Ion exchange resin, cellulose powder and the like.

In the present invention, it is desirable to use an enzyme immobilization carrier made of the material as described above and having pores that are approximately the same size as the enzyme molecule, and can be selected appropriately according to the type of enzyme to be immobilized. good.
The physical adsorption referred to here means that the enzyme is immobilized on the carrier by intermolecular attractive force such as van der Waals force or hydrophobic bond.

  For example, when the enzyme to be immobilized is a cutinase produced by Cryptococcus sp. S-2, which is a kind of yeast, the molecular size of cutinase is estimated to be 3 to 5 nm. Lebatit VP OC 1600 (manufactured by Bayer, average pore diameter 15 nm), Lebatit VP OC 1064MD (manufactured by Bayer, average pore diameter 5-10 nm), Lebatit VP OC 1163 (manufactured by Bayer, average pore diameter 0.5- 10 nm) Duolite A-568 (Rohm and Haas, pore size 15 to 25 nm), Duolite A-7 (Rohm and Haas, pore size 15 to 30 nm), and the like. Among these, it is particularly preferable to use Levacit VP OC 1600.

(Water-soluble organic solvent)
As the water-soluble organic solvent used in the present invention, water-soluble lower alcohols such as methanol, ethanol, n-propanol, and isopropanol are preferable, and methanol is particularly preferable. These water-soluble organic solvents can be used without being mixed with water, but are preferably used as a mixed solvent with water. Moreover, only 1 type may be used for a water-soluble organic solvent, and 2 or more types may be used together.

Since the above-mentioned solvent is difficult to desorb the immobilized enzyme from the carrier and other impurities can be desorbed from the enzyme-immobilized carrier, these solvents can be removed during a plurality of enzyme immobilization steps. It is considered that the enzyme can be immobilized on the carrier with high efficiency and the amount of immobilized enzyme can be improved by washing the carrier using the carrier.
However, depending on the enzyme to be immobilized, some water-soluble organic solvents have the effect of deactivating the enzyme activity, so it is important to select a water-soluble organic solvent that does not impair the enzyme activity depending on the type of enzyme. It is.

  In the present invention, when a water-soluble organic solvent is used as a mixed solvent with water, the mixing ratio of the water-soluble organic solvent and water is particularly important. The mixing ratio is not limited to a specific concentration range, and it is necessary to set the mixing ratio according to the properties of the target enzyme. The water-soluble organic solvent used for washing the immobilization carrier differs in the concentration of the organic solvent that deactivates the enzyme activity depending on the combination of the organic solvent and the enzyme used, so that the enzyme activity may be impaired depending on the target enzyme. It is important to determine a non-concentration based on the results of preliminary experiments.

  For example, in an enzyme solution used for immobilization, cutinase or lipase exists as a hydrophobic protein and is difficult to dissolve in water. On the other hand, there are many types of polysaccharides and contaminating proteins that coexist. Among contaminating proteins or polysaccharides other than the target enzyme, those having hydrophilicity and those having a molecular size larger than the pore diameter of the carrier can be removed by a washing step with water. However, hydrophobic contaminant proteins and the like cannot always be efficiently removed only by a washing step with water alone. Therefore, hydrophobic contaminating proteins can be removed by preparing an aqueous solution of an organic solvent that is more hydrophobic than water in a solvent concentration range in which the enzyme immobilized on the carrier does not dissolve, and using this to wash. For example, when washing a carrier on which cutinase or lipase is immobilized, a 1-50% aqueous methanol solution is preferred, and a 10-30% aqueous methanol solution is particularly preferred. By washing with an aqueous methanol solution having such a concentration, other impurities and the like can be detached from the immobilized carrier without effectively deactivating these enzymes and without detaching them from the carrier. Thereby, an immobilized enzyme in which cutinase or lipase is immobilized with high efficiency can be produced.

(Immobilization method)
The enzyme immobilization method of the present invention will be described below.
(Enzyme solution)
In the present invention, an enzyme solution containing a large amount of polysaccharides or contaminating proteins other than the enzyme can be suitably used for producing an immobilized enzyme.
In consideration of the long-term stability of the enzyme, it is preferable to use an enzyme solution obtained by dissolving an enzyme dry powder in a buffer solution, but a microorganism culture solution can also be used as it is. In addition, the enzyme concentration in the enzyme solution is preferably set to a concentration lower than or equal to the solubility of the enzyme in order to perform immobilization with high efficiency.
Furthermore, even if the enzyme solution does not contain a water-soluble organic solvent or contains a water-soluble organic solvent, the enzyme solution may be a water-soluble organic solvent having a lower concentration than the concentration of the water-soluble organic solvent used in the subsequent washing step. is important. For example, in the enzyme solution used for immobilization, cutinase or lipase is easily dissolved in an organic solvent, so that in the immobilization step, the presence of the organic solvent results in the enzyme being dissolved and the amount of immobilization being reduced.

  The buffer solution for dissolving the enzyme dry powder is not particularly limited, but the pH and concentration may be selected according to the enzyme characteristics so that the enzyme activity is not lost during enzyme immobilization. For example, when preparing a cutinase or lipase solution, it is preferable to use a buffer solution having a pH of 6 to 7 and a concentration of 5 to 500 mM, and in particular, a 10 to 100 mM phosphate buffer solution (pH 6.0). Is particularly preferred.

(Immobilization process)
The enzyme immobilization method in which the enzyme solution and the enzyme immobilization carrier are brought into contact with each other is not particularly limited, and can be performed according to a conventional method.
For example, the batch method in which the enzyme solution and the carrier are stirred in a plastic or glass container, or the continuous method in which the carrier is packed in a column and the enzyme solution is allowed to flow, may be frequently contacted with the enzyme molecule and the enzyme-immobilized carrier. It is important to stir, shake or flow the enzyme / carrier mixed solution so that the carrier is not damaged.

The temperature at which the enzyme is immobilized is not particularly limited as long as the enzyme does not inactivate due to heat. For example, in the case of cutinase or lipase, 5 to 30 ° C. is preferable.
In addition, the time for performing the immobilization once is not particularly limited, but it is preferable to perform the time until the enzyme immobilization reaction reaches an equilibrium state. For example, in the case of immobilization of cutinase or lipase, It is preferable to carry out over 5 to 40 hours, and it is more preferable to carry out over 10 to 24 hours.
On the other hand, in the present invention, the enzyme immobilization is performed a plurality of times, preferably three times or more, but the appropriate number of times varies depending on the type and amount of the enzyme, carrier, and contaminants used, so the amount of immobilized enzyme after each step is measured. It is more preferable to appropriately select the number of times that the amount of the immobilized enzyme reaches saturation.

(Washing process)
In the present invention, the carrier is washed at least once with a water-soluble organic solvent or an aqueous solution thereof during a plurality of enzyme immobilization steps. In order to efficiently remove impurities, the water-soluble organic solvent or It is preferable to wash each time with the aqueous solution. In addition, each cleaning step may be performed once with a water-soluble organic solvent or an aqueous solution thereof. However, one cleaning is performed by combining a cleaning with water (distilled water) and a water-soluble organic solvent or an aqueous solution thereof multiple times. It may be a process, and an optimal method may be selected as appropriate in consideration of the type of enzyme solution or carrier used.
The water-soluble organic solvent or an aqueous solution thereof is preferably a 25% methanol aqueous solution.

The cleaning method is not particularly limited, but in order to obtain a sufficient cleaning effect without damaging the carrier, it is preferable to use a method in which the cleaning liquid is inverted, stirred, shaken, fluidized, or the like.
The washing temperature at this time is not particularly limited as long as the enzyme is not deactivated by heat, but in the case of cutinase or lipase, it is preferably 5 to 30 ° C.
On the other hand, the washing time is not particularly limited as long as it has a sufficient effect of detaching other impurities from the immobilized carrier without detaching the enzyme from the carrier. For example, in the case of cutinase or lipase, Washing with an organic solvent or an aqueous solution thereof is preferably 1 to 24 hours, and more preferably 3 to 12 hours.

(Post-processing process)
The carrier on which the enzyme is immobilized may be subjected to a post-treatment according to the purpose of use of the immobilized carrier after the washing step. For example, the carrier is preferably dried by a drying method that does not impair the enzyme activity. Examples of such a drying method include cold air drying at 30 ° C. or lower, drying under reduced pressure, and the like.

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to the contents of the following examples.
Example 1
Preparation of enzyme solution A cutinase lyophilized sample containing a cutinase produced by the yeast and a polysaccharide derived from the yeast, which was obtained by culturing Cryptococcus sp-2, a kind of yeast, was prepared.
This enzyme lyophilized sample was dissolved in 50 mM phosphate buffer (pH 6.0) to a sample concentration of 100 mg / mL to prepare an enzyme solution for immobilization.

◎ 1st enzyme immobilization step As a carrier having pores having a size approximate to the molecular size of cutinase, about 134 mg of Lebatit VP OC 1600 (manufactured by Bayer) and 500 μL of the previously prepared cutinase enzyme solution in a volume of 2 mL Were mixed in a plastic tube with a lid and stirred at 15 ° C. all day and night to immobilize the enzyme on a carrier. After completion of the immobilization, the enzyme immobilization carrier and the enzyme solution subjected to the immobilization were separated by centrifugation.

◎ First washing step; water / methanol aqueous solution / water Next, the enzyme-immobilized carrier was washed five times with 500 μL of distilled water at room temperature by inversion and stirring, added with 500 μL of 25% methanol aqueous solution, and at 15 ° C. Stir for 12 hours. After stirring, the centrifugal supernatant of 25% aqueous methanol solution used for washing was removed by centrifuging at 1500 rpm for 5 minutes. Further, the separated enzyme-immobilized carrier was washed five times with 500 μL of distilled water at room temperature by the inversion / stirring method.

◎ The second enzyme immobilization step After washing the enzyme immobilization carrier with distilled water, the enzyme immobilization step in which the previously prepared enzyme solution is added again and the enzyme immobilization carrier and the enzyme solution are brought into contact with each other at 15 ° C. This was done by stirring overnight.
Finally, the enzyme-immobilized carrier was washed 5 times with 500 μL of distilled water at room temperature by inversion and stirring, and then dried at 15 ° C. to obtain an immobilized enzyme. The amount of the immobilized enzyme was measured by the following method (hereinafter the same), and as a result, it was 21.1 mg / g carrier. The results are shown in Table 1.

Determination of the amount of immobilized cutinase:
Enzyme solutions before and after enzyme immobilization were collected, and the amount of protein contained in each enzyme solution was measured with a protein assay kit manufactured by Bio-Rad. From the obtained measured value, the amount (mg) of immobilized enzyme was calculated from the following formula.
Amount of immobilized enzyme (mg) = (Amount of protein in solution before enzyme immobilization) − (Amount of protein in solution after enzyme immobilization)

(Example 2)
Second washing step: Water / aqueous methanol solution / water The carrier on which the enzyme was immobilized twice in the same manner as in Example 1 was further washed as follows.
That is, the enzyme-immobilized carrier was washed five times with 500 μL of distilled water at room temperature by inversion and stirring, added with 500 μL of 25% aqueous methanol solution, and stirred at 15 ° C. for 7 hours. After stirring, the 25% methanol aqueous solution is removed from the enzyme-immobilized support by centrifuging at 1500 rpm for 5 minutes. Further, the obtained enzyme-immobilized support is mixed with 500 μL of distilled water at room temperature by inversion and stirring. Washed twice.

◎ The third enzyme immobilization step After washing the enzyme immobilization carrier with distilled water, the enzyme solution prepared above is added again, and the enzyme immobilization carrier is brought into contact with the enzyme immobilization carrier that has been immobilized twice. The crystallization step was performed by stirring at 15 ° C. all day and night.
Finally, the enzyme-immobilized carrier was washed 5 times with 500 μL of distilled water at room temperature by inversion and stirring, and then dried at 15 ° C. to obtain an immobilized enzyme. As a result of measuring the amount of the immobilized enzyme, it was 30.6 mg per 1 g of the carrier. The results are shown in Table 1.

(Comparative Example 1)
◎ Single-time enzyme immobilization A cutinase lyophilized sample was prepared in the same procedure as in Example 1. This enzyme freeze-dried sample was dissolved in 50 mM phosphate buffer (pH 6.0) so as to have a sample concentration of 100 mg / mL to prepare an enzyme solution for immobilization.

  About 135 mg of Levacit VP OC 1600 (Bayer), which is an enzyme immobilization resin, and 500 μL of the previously prepared cutinase enzyme solution are mixed in a 2 mL-capacity plastic tube with a lid, and the enzyme and immobilization carrier are mixed. The enzyme immobilization step to be contacted was performed once by stirring at 15 ° C. all day and night.

After completion of the immobilization, the enzyme solution subjected to the immobilization was separated and removed from the enzyme immobilization carrier by centrifugation.
Next, the enzyme-immobilized carrier was washed 5 times with 500 μL of distilled water at room temperature by inversion and stirring, and then dried at 15 ° C. to obtain an immobilized enzyme. As a result of measuring the amount of the immobilized enzyme, it was 10.7 mg per 1 g of the carrier. The results are shown in Table 1.

(Comparative Example 2)
◎ No washing, enzyme immobilization three times The enzyme immobilization step was performed three times in the same manner as in Example 2 except that no washing step with distilled water / 25% aqueous methanol was inserted.
Finally, the enzyme-immobilized carrier was washed 5 times with 500 μL of distilled water at room temperature by inversion and stirring, and then dried at 15 ° C. to obtain an immobilized enzyme. As a result of measuring the amount of the immobilized enzyme, it was 27.4 mg per 1 g of the carrier. The results are shown in Table 1.

(Comparative Example 3)
◎ Water washing, enzyme immobilization three times The enzyme immobilization step was performed three times in the same manner as in Example 2 except that washing between enzyme immobilization steps was performed with 500 μL of distilled water instead of 25% methanol aqueous solution. It was.
Finally, the enzyme-immobilized carrier was washed 5 times with 500 μL of distilled water at room temperature by inversion and stirring, and then dried at 15 ° C. to obtain an immobilized enzyme. As a result of measuring the amount of the immobilized enzyme, it was 25.6 mg per 1 g of the carrier. The results are shown in Table 1.

  As shown in Table 1, by using an enzyme solution and a carrier containing impurities and immobilizing the enzyme to the carrier a plurality of times, by washing the carrier with a 25% aqueous methanol solution between the enzyme immobilization steps, More enzyme can be immobilized on the carrier than when the enzyme is immobilized once, and the enzyme immobilization process is repeated twice or three times to increase the amount of enzyme in proportion to the number of times. It was confirmed that can be immobilized on a carrier.

  On the other hand, when the washing process is not inserted as in Comparative Example 2, or when the washing process is only washing with water as in Comparative Example 3, the immobilized enzyme amount is 25% methanol aqueous solution washing. It was confirmed that it was significantly lower than

As described above, according to the method for producing an immobilized enzyme of the present invention, as an enzyme sample used for immobilization, a culture solution containing many microorganism-derived contaminants such as polysaccharides or proteins other than the enzyme to be immobilized can be used. It was confirmed that the amount immobilized on the carrier can be improved.
In addition, as described above, since the present invention can use a microorganism culture or the like as an enzyme sample, complicated and many steps such as enzyme purification can be omitted, and many immobilized enzymes can be obtained by a simple production process. Since it can be provided, it is excellent in terms of cost.

The immobilized enzyme obtained by the production method of the present invention can be subjected to various industrial enzyme reactions. In addition, since the amount of enzyme immobilized on the carrier is large, the enzyme concentration in the vicinity of the reaction point of the substrate is high, and the product can be easily separated, recovered, and reused. Can be used.

Claims (5)

Manufacture of an immobilized enzyme that immobilizes the enzyme in the enzyme solution on the carrier by performing the enzyme immobilization step that contacts the enzyme solution containing impurities and the carrier that immobilizes the enzyme by physical adsorption multiple times. A method,
A method for producing an immobilized enzyme, comprising a carrier washing step of washing a carrier at least once with a water-soluble organic solvent or an aqueous solution thereof between a plurality of enzyme immobilization steps.   The method for producing an immobilized enzyme according to claim 1, wherein the contaminant is a polysaccharide.   The method for producing an immobilized enzyme according to claim 1 or 2, wherein the water-soluble organic solvent is alcohol.   The method for producing an immobilized enzyme according to claim 3, wherein the alcohol is methanol. The method for producing an immobilized enzyme according to any one of claims 1 to 4, wherein the enzyme is lipase or cutinase.