JP2015181397A - Method of producing powdered catalyst - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of producing a powdered catalyst suitable for the industrial production of chemical products, using a recombinant Escherichia coli incorporating a gene encoding an enzyme.SOLUTION: A method of producing a powdered catalyst comprises a step in which a bacteria body-comprising liquid that comprises recombinant Escherichia coli is subjected to a spray drying process to obtain a powdered catalyst.

Description

  The present invention relates to a method for producing a powdered catalyst used when producing a chemical product.

  An enzyme is produced by a microorganism, and the microorganism is used as it is, or the enzyme is taken out from the microorganism and used as a catalyst for the production of a chemical product. If the catalyst is in a liquid state, there is a possibility of microbial contamination, which is not suitable for long-term storage. Therefore, a means for cryopreserving the catalyst-containing liquid is taken, but there is a risk that the catalyst activity is lowered when the liquid is frozen or when the frozen material is thawed. In order to solve this problem, for example, the catalyst-containing liquid is dried to a solid state. Examples of drying methods in this case include freeze drying, vacuum freeze drying, and spray drying.

  However, in order to perform freeze-drying or vacuum freeze-drying, a large-scale facility is required, and the facility investment is very expensive. Moreover, when freeze-drying or vacuum freeze-drying is employed, it is difficult to continue the entire manufacturing process. Furthermore, after freeze-drying or vacuum freeze-drying, steps such as pulverization and homogenization of the dried catalyst are required, and the manufacturing process becomes complicated.

  On the other hand, the spray drying is a method of evaporating and drying moisture by making a liquid into droplets with a atomizer and bringing the droplets into contact with a relatively high temperature drying air. When spray drying is employed, the entire manufacturing process can be continued, and workability is good at the manufacturing site.

For example, the following techniques have been proposed for spray drying of microorganisms or enzymes.
Patent Document 1 discloses a method for drying lactic acid bacteria for the purpose of increasing the viable cell rate.
Patent Document 2 discloses a method for producing a dry yeast powder having β-galactosidase activity as a method for drying bacterial cells as a catalyst.
Patent Document 3 discloses a method for producing a dry microorganism having various activities.
Patent Documents 4 and 5 disclose a method for producing a lipase powder preparation having improved lipase activity as a method for dry powdering the enzyme itself rather than the cells.
Patent Document 6 discloses a method for producing a dry powder by spray-drying an excipient and DNase and containing a biologically active form of DNase as a medicinal component.

JP 2002-17337 A International Publication No. 2012/141244 Japanese Patent Laid-Open No. 2-171184 International Publication No. 2011/68076 International Publication No. 2008/114656 Japanese National Patent Publication No. 9-509927

  If a powdered catalyst suitable for industrial production of chemicals can be obtained directly from Escherichia coli, which has an extensive track record of industrial use, industrial convenience is high.

The present invention has been made under the above circumstances.
An object of the present invention is to provide a method for producing a powdered catalyst suitable for industrial production of chemicals using recombinant Escherichia coli incorporating a gene encoding an enzyme.

Means for solving the problems are as follows.
[1] A method for producing a powdered catalyst comprising spraying and drying a cell-containing solution containing a genetically modified E. coli to obtain a powdered catalyst.
[2] The production method according to [1], wherein the content of water contained in the cell-containing liquid is 95% by mass or less based on the total mass of the cell-containing liquid.
[3] The production method according to [1], wherein the content of water contained in the cell-containing liquid is 82% by mass or more and 90% by mass or less with respect to the total mass of the cell-containing liquid.
[4] The production method according to any one of [1] to [3], wherein an activity remaining rate of the powdered catalyst is 50% or more.
[5] From the group consisting of transferase (EC.2), hydrolase (EC.3), addition / elimination enzyme (EC.4) and isomerase (EC.5) The production method according to any one of [1] to [4], wherein at least one selected enzyme is expressed.
[6] The transferase (EC.2) is at least one selected from the group consisting of a one-carbon atom transferase (EC.2.1) and a glycosyltransferase (EC.2.4). [5] The production method according to [5].
[7] The group wherein the transferase (EC.2) is composed of a hydroxymethyl group or formyl transferase (EC 2.1.2) and a pentose residue transferase (EC 2.4.2). The production method according to [5] or [6], which is at least one selected from the group consisting of:
[8] The transferase (EC.2) is at least one selected from the group consisting of glycine hydroxymethyltransferase (EC.2.1.2.1) and thymidine phosphorylase (EC 2.4.2.4). The production method according to any one of [5] to [7], which is a seed.
[9] The production method according to any one of [5] to [8], wherein the hydrolase (EC.3) is an ester hydrolase (EC.3.1).
[10] The production method according to any one of [5] to [9], wherein the hydrolase (EC.3) is a phosphate ester hydrolase (EC.3.1.3).
[11] The production method according to any one of [5] to [10], wherein the hydrolase (EC.3) is acid phosphatase (EC.3.1.3.2).
[12] The addition / desorption enzyme (EC.4) is selected from the group consisting of a carbon-carbon addition / desorption enzyme (EC. 4.1) and a carbon-oxygen addition / desorption enzyme (EC. 4.2). The production method according to any one of [5] to [11], which is at least one kind.
[13] The addition / elimination enzyme (EC.4) is selected from the group consisting of a carboxyl group addition / release enzyme (EC.4.1.1), an aldehyde group addition / release enzyme (EC.4.1.2), and dehydratases. The production method according to any one of [5] to [12], which is at least one selected from the group consisting of an enzyme (EC 4.2.1).
[14] The addition / elimination enzyme (EC.4) is benzoylformate decarboxylase (EC 4.1.1.1.7), D-threonine aldolase (EC 4.1.2.42), tryptophan synthase. Any one of [5] to [13], which is at least one selected from the group consisting of (EC 4.2.1.20) and gluconate dehydratase (EC 4.2.1.39). The manufacturing method as described in.
[15] The production method according to any one of [5] to [14], wherein the isomerase (EC.5) is an intramolecular transferase (EC.5.4).
[16] The production method according to any one of [5] to [15], wherein the isomerase (EC.5) is a phosphotransferase (EC.5.4.2).
[17] The production method according to any one of [5] to [16], wherein the isomerase (EC.5) is phosphopentomtase (EC.5.4.2.7).
[18] The production method according to any one of [1] to [17], further comprising culturing the genetically modified Escherichia coli and collecting the cultured genetically modified Escherichia coli.

  ADVANTAGE OF THE INVENTION According to this invention, the method of manufacturing the powdered catalyst suitable for the industrial production of a chemical can be provided using the recombinant Escherichia coli incorporating the gene encoding the enzyme.

It is a figure which shows the comparison result of the activity value before and behind spray-drying.

  Hereinafter, the manufacturing method of the powdered catalyst of this invention is demonstrated. The following description and examples are illustrative of the invention and are not intended to limit the scope of the invention.

In the present specification, a numerical range indicated using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
In this specification, the amount of each component in the composition is the sum of the plurality of substances present in the composition unless there is a specific indication when there are a plurality of substances corresponding to each component in the composition. Means quantity.
In this specification, the term “process” is not limited to an independent process, and is included in the term if the intended purpose of the process is achieved even when it cannot be clearly distinguished from other processes. .

<Method for producing powdered catalyst>
The method for producing a powdered catalyst of the present invention is to obtain a powdered catalyst by spray drying a cell-containing liquid containing genetically modified E. coli (hereinafter also referred to as “cell-containing liquid”) (hereinafter “ Also referred to as a “spray drying process”.
The method for producing a powdered catalyst of the present invention may include other steps as necessary. Examples of other steps include culturing the genetically modified Escherichia coli (hereinafter also referred to as “culturing step”) and collecting the cultured genetically modified Escherichia coli (hereinafter also referred to as “bacterial collecting step”). ) And the like.

[Bacteria-containing liquid]
The cell-containing solution used in the method for producing a powdered catalyst of the present invention contains genetically modified E. coli. The method for preparing the recombinant E. coli may be a known method. Examples of the method for preparing the recombinant E. coli include a method in which a gene encoding the target enzyme is incorporated into a known plasmid and incorporated into the gene of E. coli. Specifically, the method described in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), etc. may be followed. There are no particular restrictions on the type of Escherichia coli, the type of plasmid, the type of microorganisms that donate the gene, and the like used when preparing the recombinant E. coli.

  The cell-containing liquid used in the method for producing a powdered catalyst of the present invention contains water. When the present inventor diligently examined the content of water contained in the bacterial cell-containing liquid, the following knowledge was obtained.

  When the cell-containing liquid is spray-dried, the water in the cell-containing liquid evaporates due to the applied heat. While all the applied heat energy is spent on the evaporation energy of water, the heat does not act on the enzymes in the cells, so the enzyme activity is retained. However, if heat energy is applied or remains even after the water in the fungus body-containing liquid has almost evaporated, the heat acts on the enzyme in the fungus body and the activity of the enzyme decreases. Therefore, generally, the higher the content ratio of water in the bacterial cell-containing liquid to be subjected to spray drying, the less likely the enzymes in the bacterial cells are damaged during spray drying. However, when the present inventor examined, it became clear that the higher the content ratio of water contained in the bacterial cell-containing liquid, the better.

The bacterial cell-containing liquid used in the method for producing a powdered catalyst of the present invention preferably has a water content of 95% by mass or less based on the total mass of the bacterial cell-containing liquid. When the content of water contained in the microbial cell-containing liquid is within this range, the powdered catalyst obtained by spray drying can exhibit a higher activity than when the content is not so. In addition, when the content of water contained in the microbial cell-containing liquid is within this range, higher activity can be exhibited without adding a stabilizer to the microbial cell-containing solution than in the case where the content is not so.
In view of the above, the content of water contained in the bacterial cell-containing liquid is preferably 95% by mass or less, more preferably 92% by mass or less, based on the total mass of the bacterial cell-containing liquid, 90 More preferably, it is at most mass%.
The lower limit of the content of water contained in the bacterial cell-containing liquid is not particularly limited, but from the above viewpoint, it is preferably 80% by mass or more based on the total mass of the bacterial cell-containing liquid, 82 More preferably, it is more than 85 mass%, and still more preferably 85 mass% or more.
Moreover, it is preferable that the minimum of content of the water contained in a microbial cell containing liquid is 80 mass% or more from a viewpoint which contains solid content in a suitable range in order to perform spray-drying easily.
Any of the above upper limit value or lower limit value may be combined. For example, 80% by mass to 95% by mass is preferable, 82% by mass to 95% by mass is more preferable, and 82% by mass to 92% by mass is more preferable. The mass% is more preferably 90% by mass, and more preferably 85% by mass to 90% by mass.

  In this specification, the content of water contained in the bacterial cell-containing liquid is a value obtained by subtracting the solid mass from the total mass of the bacterial cell-containing liquid, and is an infrared moisture meter (for example, FD-610 manufactured by Kett Science Laboratory). ).

In the present invention, the residual activity rate of the powdered catalyst obtained by spray-drying the bacterial cell-containing liquid is preferably 50% or more, and more preferably 60% or more.
Here, the activity remaining rate of the powdered catalyst is determined by the activity value per gram of solid content (activity value before spray drying (U / g)) contained in the cell-containing liquid before spray drying and the cell-containing liquid. It is calculated from the activity value per gram of the powdered catalyst (activity value after spray drying (U / g)) measured within 24 hours after obtaining the powdered catalyst by spray drying.

More preferably, the content of water contained in the cell-containing liquid is 95% by mass or less with respect to the total mass of the cell-containing liquid, and the cell-containing liquid is spray-dried. It is preferable that the residual activity rate of the obtained powdered catalyst is 50% or more.
In the present invention, more preferably, the content of water contained in the bacterial cell-containing liquid is 82% by mass to 90% by mass with respect to the total mass of the bacterial cell-containing liquid, and the bacterial cell-containing liquid is spray-dried. It is preferable that the residual activity rate of the powdered catalyst obtained in this manner is 60% or more.
In the present invention, more preferably, the content of water contained in the bacterial cell-containing liquid is 85% by mass to 90% by mass with respect to the total mass of the bacterial cell-containing liquid, and the bacterial cell-containing liquid is spray-dried. It is preferable that the residual activity rate of the powdered catalyst obtained in this manner is 60% or more.

  So far, for example, as disclosed in Patent Documents 2 to 6, the technique of spray drying of microorganisms or enzymes has been disclosed, but for a cell-containing solution containing Escherichia coli to be subjected to spray drying, an appropriate range of water content is set. It is not easy to predict from these prior art. This will be described in detail below.

When an enzyme solution containing the enzyme itself is spray-dried, the enzyme is directly affected by the water content of the enzyme solution.
On the other hand, when a cell-containing solution containing cells expressing an enzyme is spray-dried, the enzyme is affected by the water content in the cell, and the target enzyme is not present in the other cells. It is also affected by an enzyme (for example, protease), and other enzymes present in the microbial cell are also affected by the water content in the microbial cell. In addition, the water content in the cells is affected by the water content of the cell-containing liquid. Specifically, the content of water in the cells changes due to the change in the water content of the cell-containing liquid, and for example, protease is activated by the change in the content of water in the cells. It is considered that the residual activity rate of the enzyme is greatly reduced by affecting the target enzyme.
As described above, when spray drying an enzyme solution containing the enzyme itself and when spraying a cell-containing solution containing cells expressing the enzyme, factors that should be considered for the water content Is different. That is, the appropriate water content in the liquid used for spray drying is different between the enzyme itself and the enzyme present in the cells. The degree of the influence of the water content of the liquid on the target enzyme when the cell-containing liquid is spray-dried cannot be easily predicted.

Patent Documents 4 to 6 disclose a method for dry-powdering the enzyme itself, not the cells. Therefore, it is difficult to predict an appropriate water content in the bacterial cell-containing liquid when the bacterial cell-containing liquid is spray-dried from the literature.
Moreover, Patent Documents 4 and 5 describe the content ratio of water to lipase during spray drying, but how the content ratio of water affects the activity remaining rate of lipase after spray drying. Not shown. Patent Document 6 describes the DNase concentration in the solution used for spray drying, but does not describe the water content. Patent Documents 4 to 6 do not show how the content of water relative to the enzyme during spray drying affects the residual activity rate of the enzyme after spray drying.

Patent Document 2 describes an example in which a powdered catalyst is obtained by spray drying yeasts such as Sporobolomyces singularis, Cryptococcus laurentii, Rhodotorula lactosa, Sirobasidium magnum, and Rhodotorula minuta. Patent Document 3 describes an example in which a powdered catalyst is obtained by spray drying microorganisms such as Pseudomonas putida, Fusarium lateritium, Methylophilus methylotrophus, Candida utilis, and Lactobacillus plantarum.
However, even if the powdered catalyst obtained by spray-drying the microorganisms described in Patent Documents 2 and 3 retains high activity, the same result is not always obtained in E. coli.
Furthermore, Patent Document 2 discloses the cell concentration of the cell-containing liquid used for spray drying, but does not disclose the water content. In Patent Document 2, since sugar is added as a stabilizer to the bacterial cell-containing solution, the accurate water content cannot be grasped only from the bacterial cell concentration. Patent Document 3 also discloses the cell concentration of the cell-containing liquid used for spray drying, but does not disclose the water content.
Patent Documents 2 and 3 do not discuss how the water content affects the catalyst activity remaining rate after spray drying.

  As described above, it has not been easy to predict an appropriate range of water content for the bacterial cell-containing liquid containing Escherichia coli to be subjected to spray drying from the prior art.

Hereinafter, description of the microbial cell containing liquid in this invention is continued.
The cell-containing liquid in the present invention may contain a stabilizer. Various compounds can be used as the stabilizer. Specifically, for example, sugars such as glucose, lactose, fructose, starch, cyclodextrin; amino acids such as glycine, alanine, and glutamic acid; nucleic acids such as thymidine, guanylic acid, and adenosine triphosphate; citric acid, fumaric acid, Organic acids such as lactic acid; metals such as magnesium, calcium and sodium; mixtures obtained by enzymatic treatment of soy protein hydrolyzate, wheat gluten hydrolyzate, yeast extract and the like; enzyme substrates exhibiting the desired activity; Products, coenzymes, prosthetic groups; and the like.

The pH of the bacterial cell-containing solution in the present invention is not particularly limited as long as the catalytic activity of the target enzyme is not excessively lost, and may be a pH range in which the enzyme is not denatured. Specifically, the pH is preferably in the range of 4 to 10, and preferably exhibits a pH at which the catalytic activity of the target enzyme is not lost.
Some enzymes are stable in the acidic region and others are stable in the neutral or alkaline region. What is necessary is just to select pH suitable for each enzyme, and to adjust pH of a microbial cell containing liquid using a pH adjuster etc. The kind of pH adjuster is not particularly limited.
In this specification, the pH of the bacterial cell-containing liquid is measured with a temperature of the bacterial cell-containing liquid at 25 ° C., for example, using a pH meter (D-52 manufactured by Horiba, Ltd.).

[Enzymes contained in powdered catalyst]
In the present invention, the enzyme contained in the powdered catalyst is derived from genetically modified Escherichia coli contained in the cell-containing solution. In general, enzymes are EC. 1 to EC. It is classified into 6 of 6. EC. 1 is an oxidoreductase, EC. 2 is a transferase, EC. 3 is a hydrolase, EC. 4 is an addition / elimination enzyme, EC. 5 is an isomerase, EC. 6 is a synthetic enzyme.

Conventionally, it has been studied how much the catalytic activity of an enzyme is maintained when a microorganism is spray-dried. For example, Patent Document 3 discloses an enzyme activity retention rate when a microorganism is spray-dried, and the enzyme activity retention rate in a glycolysis system and an amino acid biosynthesis system when Lactobacillus plantarum is spray-dried is 100%. Have been described.
However, when the microorganism used for spray drying is Escherichia coli, it is not easy to predict the degree of retention of the catalytic activity of the enzyme from the related art. This will be described in detail below.

As shown in Reference Example 1 to be described later, for propanediol oxidoreductase (EC.1.1.1.17), a powdered catalyst was obtained by spray drying a cell-containing solution containing genetically modified E. coli, As a result, the activity of the powdered catalyst was greatly reduced.
Same classification as propanediol oxidoreductase EC. An enzyme belonging to 1 is glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate: NAD + oxidoreductase) (EC 1.2.1.12). This enzyme is an enzyme involved in glycolysis. Ascertained from the glycolytic enzyme activity retention shown in Patent Document 3, EC. 1 and the EC. Obtained in Reference Example 1 described later by the present inventor. It is difficult to predict the degree of retention of catalytic activity from the prior art when the cell-containing liquid containing Escherichia coli is spray-dried. It shows.

  Patent Document 3 also discloses EC. D-2-haloalkanoic acid halide hydrolase (Pseudomonasputida) and amidase (Methylophilus methylotrophus), EC. An example is described in which microorganisms originally retaining an enzyme such as cyanide hydratase (Fusarium lateritium) and pyruvate decarboxylase (Candida utilis) belonging to 4 are spray-dried. However, EC. 3 and EC. Even if the activity retention rate of the enzyme belonging to 4 is shown, the EC. As with the enzyme belonging to No. 1, it is difficult to predict how much catalytic activity will remain in the recombinant E. coli.

  As a result of intensive studies by the present inventors, when the bacterial cell-containing liquid is spray-dried, the catalyst activity remaining rate after spray-drying varies greatly depending on the type of microorganism, and the combination of the type of enzyme and the type of microorganism The knowledge that it depends also was obtained. Since the activity remaining rate of the powdered catalyst can vary depending on the enzyme type of the enzyme species to be spray-dried, the enzyme applied to the present invention is EC. 2-EC. 6 is preferred, EC. 2-EC. 5 is more preferable.

The oxidoreductase (EC.1) is an enzyme that exchanges electrons with an electron donor or electron acceptor, and most of them involve a coenzyme in the reaction. When the cell-containing liquid containing oxidoreductase is spray-dried, as shown in Reference Example 1 described later, the degree of activity of the powdered catalyst obtained by spray-drying is excessively lost compared to other enzymes. Is called. Therefore, EC. Enzymes classified as 1 are not suitable for a method for producing a powdered catalyst by spray drying.
Specific examples of the oxidoreductase (EC.1) include alcohol dehydrogenase (EC.1.1.1.1), propanediol oxidoreductase (EC.1.1.1.17), glucose oxidase (EC 1.1.3.4), glutamate dehydrogenase (EC 1.4.1.2), cyanocobalamin reductase (EC 1.16.1.6) and the like.

Enzymes suitable for the production method of the powdered catalyst of the present invention include transferase (EC.2), hydrolase (EC.3), addition / elimination enzyme (EC.4), and isomerase (EC.5). And at least one selected from the group consisting of a synthetic enzyme (EC.6), more preferably a transferase (EC.2), a hydrolase (EC.3), and an addition / desorption enzyme (EC.4). And at least one selected from the group consisting of isomerase (EC.5).
It is sufficient that at least one of these enzymes is expressed in the genetically modified Escherichia coli in the present invention, and two or more enzymes may be expressed.

As the transferase (EC.2), one-carbon atom transferase (EC.2.1) and glycosyltransferase (EC.2.4) are preferable. More preferred are hydroxymethyl group or formyltransferase (EC 2.1.2), pentose residue transferase (EC 2.4.2), and more preferred is glycine hydroxymethyl transferase (EC. 2.1.2.1), thymidine phosphorylase (EC 2.4.2.4).
It is sufficient that at least one of these transferases is expressed in the recombinant E. coli, and two or more thereof may be expressed in the recombinant E. coli.

  The hydrolase (EC.3) is preferably an ester hydrolase (EC.3.1), more preferably a phosphate ester hydrolase (EC.3.1.3), and still more preferably an acidic enzyme. Phosphatase (EC 3.3.1.3.2).

The addition / desorption enzyme (EC.4) is preferably a carbon-carbon addition / desorption enzyme (EC.4.1) or a carbon-oxygen addition / desorption enzyme (EC.4.2), more preferably a carboxyl group addition. Desorption enzyme (EC 4.1.1), aldehyde group addition / desorption enzyme (EC 4.1.2), dehydratase enzyme (EC 4.2.1), more preferably benzoylform Mate decarboxylase (EC 4.1.1.1.7), D-threonine aldolase (EC 4.1.2.42), tryptophan synthase (EC 4.2.1.20), gluconate dehydratase (EC 4.2.1.39).
It is sufficient that at least one of these addition / elimination enzymes is expressed in the recombinant E. coli, and two or more of them may be expressed in the recombinant E. coli.

  The isomerase (EC.5) is preferably an intramolecular transferase (EC.5.4), more preferably a phosphotransferase (EC.5.4.2), and even more preferably a phosphopent. Mutase (EC 5.4.2.7).

  Examples of the synthase (EC.6) include acetyl CoA synthetase (EC.6.2.1.1), glutamate synthetase (EC.6.3.1.2), and DNA ligase (EC.6.5. 1) etc. are mentioned.

[Spray drying process]
In the spray drying step of the present invention, the method for preparing a cell-containing solution containing genetically modified Escherichia coli is not particularly limited.
For example, a culture solution obtained in the culture step described later can be used as the bacterial cell-containing solution. In addition, at least one selected from the group consisting of water; buffer; medium; aqueous solution containing coenzyme and / or prosthetic metal; aqueous solution containing various compounds is added to the cells obtained in the bacterial collection step described later. It may be prepared by
When preparing the microbial cell-containing liquid, it is preferable to prepare such that the optical density (OD value) of the microbial cell-containing liquid is 80 to 500. Thereby, the water content suitable for spray drying can be obtained, which is preferable. The optical density (OD value) of the bacterial cell-containing liquid can be measured with a spectrophotometer (for example, UV-1800 manufactured by Shimadzu Corporation).

In the spray-drying step of the present invention, the method for obtaining a powdered catalyst by spray-drying the cell-containing liquid is not particularly limited. For example, by spray-drying the cell-containing liquid with a spray dryer or the like, A powdered catalyst for the body-containing liquid can be obtained.
Any type of spray dryer may be used as long as it can dry the cell-containing liquid by spraying.
Examples of the spraying method of the bacterial cell-containing liquid include a one-fluid nozzle method, a two-fluid nozzle method, a gas-saving atomizing nozzle method, a twin jet nozzle method, a rotary atomizer method, and the like. Any method may be used if possible. Among these, in order to spray-dry a liquid containing microparticles of E. coli, it is preferable to employ a two-fluid nozzle method or a rotary atomizer method.

In the spray-drying process in the present invention, the amount of dry air of the spray dryer when spray-drying the cell-containing liquid is not particularly limited as long as it is an amount of dry air that can spray-dry the cell-containing liquid. Since the processing capacity varies depending on the size and type of the spray dryer, the dry air amount of the spray dryer may be determined according to the size and type of the spray dryer. For example EYELA SD-1000 as a spray dryer when using (Tokyo Rika Kikai Co., Ltd.) as the dry air of the spray dryer, particularly preferably 0.3m ³ /min~0.75m ³ / min, and more preferably 0.5m ³ /min~0.75m ³ / min.

In the spray-drying process of the present invention, the inlet temperature of the spray dryer when spray-drying the cell-containing liquid is such that the cell-containing liquid can be spray-dried and the catalytic activity of the target enzyme is excessively lost. There is no particular limitation as long as the temperature is not changed. Specifically, the inlet temperature of the spray dryer is preferably 100 ° C to 250 ° C, more preferably 130 ° C to 200 ° C.
Here, that the catalytic activity of the target enzyme is not excessively lost means, for example, that the catalytic activity of the enzyme after spray drying is 50% or more compared to the catalytic activity of the enzyme before spray drying. means.

  In the spray-drying process of the present invention, the outlet temperature of the spray dryer when spray-drying the cell-containing liquid is such that the cell-containing liquid can be spray-dried and the catalytic activity of the target enzyme is excessively lost. There is no particular limitation as long as the temperature is not changed. Specifically, the outlet temperature of the spray dryer is preferably 50 ° C to 150 ° C, more preferably 70 ° C to 120 ° C.

In the spray drying step of the present invention, the liquid feeding speed when spray-drying the cell-containing liquid is not particularly limited as long as the cell-containing liquid can be dried and a powdered catalyst can be produced.
Since the treatment capacity varies depending on the size, type, etc. of the spray dryer, the feeding speed of the cell-containing liquid may be determined according to the size, type, etc. of the spray dryer.
Specifically, it is preferable to adopt a liquid feeding speed of 50% to 100% of the capacity described in the equipment specification attached to each spray dryer, and more preferably, a liquid feeding speed of 70% to 90%. Can be adopted. For example, when EYELA SD-1000 (manufactured by Tokyo Science Instruments Co., Ltd.) or the like is used as a spray dryer, it is preferable to adjust the feeding speed of the cell-containing liquid to 300 g / hr to 1500 g / hr, and 500 g / hr. It is more preferable to adjust to -1200 g / hr, and it is still more preferable to adjust to 700 g / hr-1000 g / hr.

In the spray-drying step in the present invention, the spray-drying step is performed such that the water content of the powdered catalyst is 2% by mass to 20% by mass (more preferably 2% by mass to 15% by mass, More preferably, it is preferably carried out until it is in the range of 3% by mass to 12% by mass. Thereby, the activity remaining rate of the obtained powdered catalyst can be maintained in a preferable range.
In this specification, the water content of the powdered catalyst is a value obtained by subtracting the solid mass from the total mass of the powdered catalyst, and can be measured by an infrared moisture meter (for example, FD-610 manufactured by Kett Science Laboratory). .

  When the spraying method of the spray dryer used is a two-fluid nozzle method, the spraying pressure at the time of spray drying affects the volume average particle diameter of the powdered catalyst obtained by spray drying. Therefore, when using the two-fluid nozzle method, it is preferable to adjust the spray pressure when spray-drying the bacterial cell-containing liquid to 150 kPa to 250 kPa, more preferably 150 kPa to 220 kPa, and 180 kPa to More preferably, the pressure is adjusted to 220 kPa.

  When the spraying method of the spray dryer used is a rotary atomizer method, the rotation speed of the atomizer affects the volume average particle diameter of the powdered catalyst obtained by spray drying. Therefore, when using a rotary atomizer system, it is preferable to set the rotation speed of a rotary atomizer to 15000 rpm to 30000 rpm, for example, and more preferably to 20000 rpm to 25000 rpm.

The average volume particle diameter of the powdered catalyst obtained by the spray drying step is preferably 5 nm to 100 nm (more preferably 10 nm to 80 nm, still more preferably 10 nm to 60 nm). Thereby, the solubility with respect to the solvent of a powdered catalyst is good, and it can exhibit a catalyst ability.
In this specification, the average volume particle diameter of the powdered catalyst can be measured by a particle size distribution measuring apparatus (for example, Microtrac MT-3200 manufactured by Nikkiso Co., Ltd.).

[Culture process]
The method for producing a powdered catalyst of the present invention may further include a culture step of culturing the genetically modified Escherichia coli.
The culture process may be determined according to the type of genetically modified Escherichia coli used in the method for producing a powdered catalyst of the present invention. For the culture, for example, pre-culture is performed at a culture temperature of 25 ° C. to 37 ° C. with stirring for 6 to 24 hours, and main culture is performed at a culture temperature of 25 ° C. to 37 ° C. with stirring for 20 to 50 hours. The medium is not particularly limited, and examples thereof include an LB medium, an NB medium, a MacConkey medium, a medium in which an inorganic salt is added to peptone, and the like.

  In the method for producing a powdered catalyst of the present invention, the culture solution obtained by the culturing step may be used as the cell-containing solution. Moreover, you may perform the microbe collection process mentioned later after performing a culture | cultivation process.

[Bacteria collection process]
The method for producing a powdered catalyst of the present invention may further include a collection step for collecting the cultured recombinant E. coli.
The method of collecting bacteria is not particularly limited. Examples of the method of collecting bacteria include a method of collecting bacteria by putting the culture solution obtained in the culturing step into a bottle and centrifuging for about 20 minutes using a centrifuge. Moreover, the method of collecting bacteria by centrifuging a culture solution with a continuous centrifuge is also mentioned.

  In the method for producing a powdered catalyst of the present invention, the cells obtained in the collection process are mixed with water; buffer; medium; aqueous solution containing coenzyme and / or prosthetic metal; aqueous solution containing various compounds. You may prepare a microbial cell containing liquid by adding at least 1 sort (s) chosen from more.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

<Reference Example 1>
[Construction of E. coli expressing propanediol oxidoreductase]
Based on the known information (GenBank accession number M31059) of the propanediol oxidoreductase gene of Escherichia coli, the oligonucleotides shown in SEQ ID NO: 1 and SEQ ID NO: 2 were synthesized. Using the genomic DNA of Escherichia coli MG1655 strain (ATCC70096) as a template, using the oligonucleotides shown in SEQ ID NO: 1 and SEQ ID NO: 2 as primers, amplification by PCR method, and digesting the resulting DNA fragment with restriction enzymes EcoI and HindIII As a result, a fucO fragment of about 1.2 kbp was obtained.
The fucO fragment and a fragment obtained by digesting plasmid pUC18 (manufactured by Toyobo Co., Ltd.) with restriction enzymes EcoRI and HindIII were ligated using ligase and then transformed into Escherichia coli DH5α strain (manufactured by Toyobo Co., Ltd.). Thus, a transformant that grew on an LB agar plate containing 50 μg / mL of ampicillin was obtained.
The obtained transformant was cultured overnight at 37 ° C. in an LB liquid medium containing 50 μg / mL of ampicillin. A plasmid was recovered from the obtained microbial cells, and this plasmid was designated as pGAPfucO.
The Escherichia coli MG1655 strain was transformed with the plasmid pGAPfucO and cultured overnight at 37 ° C. on an LB agar plate containing 50 μg / mL of ampicillin to obtain an MG1655 / pGAPfucO strain.

[Culture of Escherichia coli expressing propanediol oxidoreductase]
The MG1655 / pGAPfucO strain was inoculated into 25 mL of LB Broth, Miller culture solution (Difco244620) in an Erlenmeyer flask and cultured overnight at a temperature of 35 ° C. and a stirring speed of 120 rpm. The total amount of this preculture was transferred to a 1 L culture device (BMJ-01 manufactured by ABLE) containing 475 g of a medium having the composition shown below, and cultured. Culturing was performed under atmospheric pressure with an aeration rate of 0.5 L / min, a stirring speed of 800 rpm, a culture temperature of 35 ° C., and a pH of 7.2 (adjusted with an NH ₃ aqueous solution). After the initial glucose was completely depleted, 40 g total glucose was fed over the remaining culture time at a variable rate such that the glucose concentration in the medium was less than 0.1 g / L.

-Medium composition-
Polypeptone: 7g / L
Fe ₂ SO ₄ : 0.09 g / L
K ₂ HPO ₄ : 2 g / L
KH ₂ PO ₄ : 2 g / L
MgSO ₄ · 7H ₂ O: 2 g / L
(NH ₄ ) ₂ SO ₄ : 1.5 g / L
Glucose: 30 g / L

[Preparation of cell-containing liquid]
Cells were collected 24 hours after the start of culture by centrifugation (8000 rpm, 20 minutes). Water was added to the collected cells and three types of cell-containing liquids having different water contents were prepared (Samples 1 to 3). The water content of the prepared bacterial cell-containing liquid was measured with an infrared moisture meter (FD-610, manufactured by Kett Science Laboratory). The measurement results are shown in Table 1.

[Spray drying of E. coli expressing propanediol oxidoreductase]
The bacterial cell-containing liquids of Samples 1 to 3 were spray-dried with a spray dryer EYELA SD-1000 (manufactured by Tokyo Science Instruments). The conditions for spray drying are as follows.

-Spray drying conditions-
Spray method: two-fluid nozzle drying air ^{quantity: 0.66m 3 /min~0.81m} 3 / min
Spray pressure: 190 kPa
Inlet temperature: 140 ° C to 146 ° C
Liquid feeding speed: 614 g / hr to 638 g / hr

  The water content of the obtained powdered bacterial cells (powdered catalyst) was measured with an infrared moisture meter (FD-610, manufactured by Kett Science Laboratory). The measurement results are shown in Table 1.

[Comparison of activity values before and after spray drying]
The bacterial cell-containing solutions of Samples 1 to 3 were each centrifuged at 8000 rpm for 20 minutes, and the collected bacterial cells were washed twice with 50 mM phosphate buffer (pH 7.4). After suspending in the same buffer, the mixture was crushed with an ultrasonic crusher, and the supernatant obtained by centrifugation was used as an enzyme solution for activity measurement.
In addition, the powdered cells (powdered catalyst) after spray drying of samples 1 to 3 were suspended in 50 mM phosphate buffer (pH 7.4), crushed with an ultrasonic crusher, and obtained by centrifugation. Kiyoshi was used as an enzyme solution for activity measurement.

An aqueous solution containing 250 mM ethylene glycol, 2 mM NAD, and 100 mM glycine buffer (pH 9.5) was set in a spectrophotometer (BioSpec-1600 manufactured by Shimadzu Corporation), the temperature was kept at 30 ° C. with CPS-CONTROLLER, and 30 μL of enzyme solution was added. After addition, the change in absorbance of the OD value at 340 nm was measured.
For the activity value, the amount of dry cells that can change the value of the OD value at 340 nm by 1 per minute was defined as 1 U.
The measurement results are shown in Table 2 and FIG. In FIG. 1, the results for samples 1 to 3 are shown as PO bacteria.

<Example 1>
[Construction of E. coli expressing glycine hydroxymethyltransferase]
Based on the known information (GenBank accession number AP009048) of the glycine hydroxymethyltransferase gene of Escherichia coli, oligonucleotides shown in SEQ ID NO: 3 and SEQ ID NO: 4 were synthesized. Using the genomic DNA of Escherichia coli W3110 strain (ATCC27325) as a template, using the oligonucleotides shown in SEQ ID NO: 3 and SEQ ID NO: 4 as primers, the DNA fragment is amplified by PCR, and the resulting DNA fragment is digested with restriction enzymes EcoRI and HindIII As a result, a glyA fragment of about 1.3 kbp was obtained.
The ligA fragment and a fragment obtained by digesting plasmid pUC18 (manufactured by Toyobo Co., Ltd.) with restriction enzymes EcoRI and HindIII were ligated using ligase, and then transformed into Escherichia coli DH5α strain (manufactured by Toyobo Co., Ltd.). Thus, a transformant that grew on an LB agar plate containing 50 μg / mL of ampicillin was obtained.
The obtained transformant was cultured overnight at 37 ° C. in an LB liquid medium containing 50 μg / mL of ampicillin. A plasmid was recovered from the obtained bacterial cells, and this plasmid was designated as pUCglyA.
Escherichia coli W3110 was transformed with the plasmid pUCglyA and cultured overnight at 37 ° C. on an LB agar plate containing 50 μg / mL of ampicillin to obtain a W3110 / pUCglyA strain.

[Culture of Escherichia coli expressing glycine hydroxymethyltransferase]
The W3110 / pUCglyA strain was inoculated into 200 mL of LB Broth, Miller culture medium (Difco 244620) in an Erlenmeyer flask and cultured overnight at a temperature of 33 ° C. and a stirring speed of 120 rpm. The total amount of this preculture was transferred to a 10 L culture apparatus (BMS-10PI manufactured by ABLE) containing 5 L of a medium having the composition shown below and cultured. Culturing was performed under atmospheric pressure with an aeration rate of 0.5 L / min, a stirring speed of 600 rpm, a culture temperature of 28 ° C., and a pH of 7.3 (adjusted with an NH ₃ aqueous solution). After the initial glucose was completely depleted, glucose was fed at a variable rate so that the glucose concentration in the medium was less than 0.1 g / L over the remaining culture time.

-Medium composition-
Polypeptone: 7g / L
Fe ₂ SO ₄ : 0.09 g / L
K ₂ HPO ₄ : 2 g / L
KH ₂ PO ₄ : 2 g / L
MgSO ₄ · 7H ₂ O: 2 g / L
(NH ₄ ) ₂ SO ₄ : 1.5 g / L
Glucose: 15 g / L

[Preparation of cell-containing liquid]
48 hours after the start of culture, the cells were collected by centrifugation (8000 rpm, 20 minutes). Water was added to the collected cells, and four types of cell-containing liquids having different water contents were prepared (samples 4 to 7). The water content of the prepared bacterial cell-containing liquid was measured with an infrared moisture meter (FD-610, manufactured by Kett Science Laboratory). Table 3 shows the measurement results.

[Spray drying of Escherichia coli expressing glycine hydroxymethyltransferase]
The cell-containing liquids of Samples 4 to 7 were spray-dried with a spray dryer in the same manner as the spray-drying in Reference Example 1 to obtain powdered cells (powdered catalyst). The water content of the obtained powdered bacterial cells (powdered catalyst) was measured with an infrared moisture meter (FD-610, manufactured by Kett Science Laboratory). Table 3 shows the measurement results.

[Comparison of activity values before and after spray drying]
The enzyme solution before spray drying was prepared in the same manner as in Reference Example 1. The enzyme solution after spray drying was prepared in the same manner as in Reference Example 1 except that 10 mM Tris buffer (pH 7.5) was used.

An aqueous solution containing 500 mM glycine, 100 mM phosphate buffer (pH 7.3) and 400 mM tetrahydrofolic acid was placed in a screw tube, kept at 45 ° C. in a water bath, and 0.1 mL of enzyme solution was added to initiate the reaction. After 10 minutes, 1/4 amount of 15% (w / w) trichloroacetic acid was added to stop the reaction, and the amount of L-serine produced was analyzed by HPLC.
For the activity value, the amount of dry cells capable of producing 1 μmol of L-serine per minute was defined as 1 U.
The measurement results are shown in Table 4 and FIG. In FIG. 1, the results for samples 4 to 7 are shown as GT bacteria.

<Example 2>
[Construction of Escherichia coli expressing various enzymes]
In the same manner as in the construction of the expression Escherichia coli in Example 1, Escherichia coli expressing various enzymes was prepared. Details are shown in Table 5. Since gluconate dehydratase is not registered in GenBank, the oligonucleotides shown in SEQ ID NO: 13 and SEQ ID NO: 14 were synthesized based on the sequence information described in Japanese Patent No. 4391328.

[Culture of expressed Escherichia coli and preparation of cell-containing solution]
E. coli expressing each enzyme was cultured in the same manner as the E. coli culture in Example 1, and a cell-containing solution as shown in Table 6 was prepared from the culture solution in the same manner as in Example 1 (Samples 8 to 35). . The water content of the prepared bacterial cell-containing liquid was measured with an infrared moisture meter (FD-610, manufactured by Kett Science Laboratory). Table 6 shows the measurement results.

[Spray drying with two-fluid nozzle method]
The bacterial cell-containing liquids of Samples 8 to 35 were spray-dried with a spray dryer in the same manner as the spray-drying in Reference Example 1 to obtain powdered bacterial cells (powdered catalyst). The water content of the obtained powdered bacterial cells (powdered catalyst) was measured with an infrared moisture meter (FD-610, manufactured by Kett Science Laboratory). Table 6 shows the measurement results.

[Comparison of activity values before and after spray drying]
The enzyme solution was prepared in the same manner as in Example 1. The method for measuring the activity of each enzyme and the definition of the activity value are as follows.

-Thymidine phosphorylase-
An aqueous solution containing 100 mM thymine, 100 mM 2-deoxyribose-1-phosphate (dR1P) ammonium salt and 180 mM magnesium hydroxide is placed in a screw tube, kept at 40 ° C. in a water bath, and 0.2 mL of enzyme solution is added to react. Started. After 30 minutes, an equal amount of 1N aqueous sodium hydroxide solution was added to stop the reaction, and the amount of thymidine produced was analyzed by HPLC. For the activity value, the amount of dry cells capable of producing 1 μmol of thymidine per minute was defined as 1 U.

-Acid phosphatase-
An aqueous solution containing 300 mM sodium pyrophosphate and 600 mM 2-deoxyribose was placed in a screw tube, kept at 10 ° C. in a water bath, and 1 mL of enzyme solution was added to initiate the reaction. After 30 minutes, 3 times the amount of 1N hydrochloric acid was added to stop the reaction, and the amount of 2-deoxyribose-5-phosphate produced was analyzed by HPLC. The activity value was defined as 1 U as the amount of dry cells capable of producing 1 μmol of 2-deoxyribose-5-phosphate per minute.

-D-threonine aldolase-
An aqueous solution containing 2M glycine, 500 mM formalin, 0.1 mM pyridoxal phosphate, 10 mM magnesium chloride is placed in a screw tube, adjusted to pH 6.6 with an aqueous sodium hydroxide solution, kept at 35 ° C. in a water bath, and 0.5 mL of enzyme solution. Was added to start the reaction. After 20 minutes, an equal amount of 2N hydrochloric acid was added to stop the reaction, and the amount of D-serine produced was analyzed by HPLC. For the activity value, the amount of dry cells capable of producing 1 μmol of D-serine per minute was defined as 1 U.

-Benzoylformate decarboxylase-
An aqueous solution containing 200 mM 2-keto-3-deoxygluconic acid, 20 mM phosphate buffer (pH 7.0), 5 mM magnesium chloride, 1 mM thiamine pyrophosphate is placed in a screw tube, kept at 50 ° C. in a water bath, and 1 mL of enzyme solution is added. The reaction was started by addition. After 60 minutes, 1/10 amount of 1N hydrochloric acid was added to stop the reaction, and the amount of 2-deoxyribose produced was analyzed by HPLC. For the activity value, the amount of dry cells capable of producing 1 μmol of 2-deoxyribose per minute was defined as 1 U.

-Gluconic acid dehydratase-
An aqueous solution containing 40 mM sodium gluconate, 100 mM Tris buffer (pH 8.5) and 10 mM magnesium chloride was placed in a screw tube, kept at 37 ° C. in a water bath, and 0.5 mL of enzyme solution was added to initiate the reaction. Ten minutes later, 1/5 hydrochloric acid 1N hydrochloric acid was added to stop the reaction, and the amount of 2-keto-3-deoxygluconic acid produced was analyzed by HPLC. The activity value was defined as 1 U as the amount of dry cells capable of producing 1 μmol of 2-keto-3-deoxygluconic acid per minute.

-Tryptophan synthase-
An aqueous solution containing 750 mM L-serine, 3 mM indole and 0.05 mM pyridoxal phosphate was placed in a screw tube, kept at 45 ° C. in a water bath, and 0.05 mL of enzyme solution was added to initiate the reaction. Ten minutes later, 1/5 amount of 1N phosphoric acid was added to stop the reaction, and the amount of produced tryptophan was analyzed by HPLC. For the activity value, the amount of dry cells capable of producing 1 μmol of tryptophan per minute was defined as 1 U.

-Phosphopentomase-
A liquid containing 100 mM thymine, 100 mM 2-deoxyribose-5-phosphate, 180 mM magnesium hydroxide, 50 mM manganese chloride, 0.45% (w / w) thymidine phosphorylase-expressing microbial cells was placed in a screw tube, and 40 minutes in a water bath. The reaction was started by adding 0.45 mL of the enzyme solution while maintaining the temperature. After 30 minutes, an equal amount of 1N aqueous sodium hydroxide solution was added to stop the reaction, and the amount of thymidine produced was analyzed by HPLC. For the activity value, the amount of dry cells capable of producing 1 μmol of thymidine per minute was defined as 1 U.

  The measurement results are shown in Table 7 and FIG. In FIG. 1, the results for samples 8-11 are TP bacteria, the results for samples 12-15 are AP bacteria, the results for samples 16-19 are DT bacteria, the results for samples 20-23 are BD bacteria, and the sample 24 The results for -27 are shown as GD bacteria, the results for samples 28-31 as TS bacteria, and the results for samples 32-35 as PM bacteria.

[Spray drying with rotary atomizer]
The bacterial cell-containing liquid prepared in the same manner as Sample 29 was spray-dried with a spray dryer L-8i (manufactured by Okawara Chemical Co., Ltd.). The conditions for spray drying are as follows.

-Spray drying conditions-
Spraying method: Rotary atomizer Rotation speed: 20000 rpm
Inlet temperature: 150 ° C
Liquid feeding speed: 1.1 kg / hr

  The water content of the obtained powdered bacterial cell (powdered catalyst) was measured with an infrared moisture meter (FD-610 manufactured by Kett Science Laboratory), and as a result, it was 7.5 with respect to the total mass of the powdered bacterial cell. It was mass%. Table 8 shows the results of measuring the activity of tryptophan synthase in the same manner as described above.

Claims (18)

  A method for producing a powdered catalyst comprising spraying and drying a cell-containing solution containing genetically modified Escherichia coli to obtain a powdered catalyst.   The manufacturing method of Claim 1 whose content of the water contained in the said microbial cell containing liquid is 95 mass% or less with respect to the total mass of this microbial cell containing liquid.   The manufacturing method of Claim 1 whose content of the water contained in the said microbial cell containing liquid is 82 to 90 mass% with respect to the total mass of this microbial cell containing liquid.   The production method according to any one of claims 1 to 3, wherein an activity remaining rate of the powdered catalyst is 50% or more.   At least selected from the group consisting of a transferase (EC.2), a hydrolase (EC.3), an addition / elimination enzyme (EC.4) and an isomerase (EC.5) in the genetically modified Escherichia coli. The production method according to any one of claims 1 to 4, wherein one kind of enzyme is expressed.   The transferase (EC.2) is at least one selected from the group consisting of a single carbon atom transferase (EC.2.1) and a glycosyltransferase (EC.2.4). The manufacturing method as described in.   The transferase (EC.2) is selected from the group consisting of a hydroxymethyl group or formyltransferase (EC 2.1.2) and a pentose residue transferase (EC 2.4.2). The manufacturing method of Claim 5 or Claim 6 which is at least 1 sort (s).   The transferase (EC.2) is at least one selected from the group consisting of glycine hydroxymethyltransferase (EC.2.1.2.1) and thymidine phosphorylase (EC 2.4.2.4). The manufacturing method of any one of Claims 5-7.   The production method according to any one of claims 5 to 8, wherein the hydrolase (EC.3) is an ester hydrolase (EC.3.1).   The manufacturing method according to any one of claims 5 to 9, wherein the hydrolase (EC.3) is a phosphate ester hydrolase (EC.3.1.3).   The production method according to any one of claims 5 to 10, wherein the hydrolase (EC.3) is acid phosphatase (EC.3.1.3.2).   The adductase (EC.4) is at least one selected from the group consisting of a carbon-carbon adductase (EC.4.1) and a carbon-oxygen adductase (EC.4.2). The manufacturing method of any one of Claims 5-11 which is these.   The addition / elimination enzyme (EC.4) comprises a carboxyl group addition / release enzyme (EC.4.1.1), an aldehyde group addition / release enzyme (EC.4.1.2) and a dehydratase enzyme (EC). The manufacturing method according to any one of claims 5 to 12, which is at least one selected from the group consisting of.   The addition / elimination enzyme (EC.4) is benzoylformate decarboxylase (EC 4.1.1.1.7), D-threonine aldolase (EC 4.1.2.42), tryptophan synthase (EC. The method according to any one of claims 5 to 13, which is at least one selected from the group consisting of 4.2.20) and gluconate dehydratase (EC 4.2.1.39). Production method.   The production method according to any one of claims 5 to 14, wherein the isomerase (EC.5) is an intramolecular transferase (EC.5.4).   The production method according to any one of claims 5 to 15, wherein the isomerase (EC.5) is a phosphotransferase (EC.5.4.2).   The production method according to any one of claims 5 to 16, wherein the isomerase (EC.5) is a phosphopentomtase (EC.5.4.2.7).   The production method according to any one of claims 1 to 17, further comprising culturing the genetically modified Escherichia coli and collecting the cultured genetically modified Escherichia coli.